Dendrimer compositions and methods for drug delivery to the eye

ABSTRACT

Dendrimer compositions and methods for the treatment of one or more inflammatory and/or angiogenic diseases and/or disorders of the eye include hydroxyl-terminated dendrimers complexed or conjugated with one or more active agents for the treatment or alleviation of one or more symptoms of the diseases of the eye, and/or for diagnosing the diseases and/or disorders of the eye. The dendrimers may include one or more ethylene diamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated generation-4, 5, 6, 7, 8, 9, or 10 dendrimers. The active agents may be VEGFR tyrosine kinase inhibitors including sunitinib or analogues thereof. Preferably, the compositions are suitable for administration via a systemic route to target activated microglia/macrophages in retina/choroid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/943,724, filed Dec. 4, 2019, U.S. Provisional Application No.63/021,023, filed May 6, 2020, and U.S. Provisional Application No.63/108,234, filed Oct. 30, 2020, which are incorporated by referenceherein in their entirety.

FIELD OF THE INVENTION

The invention is generally in the field of drug delivery, and inparticular, a method of delivering drugs selectively to activated immunecells within the eye and surrounding tissue.

BACKGROUND OF THE INVENTION

The development of neuroinflammatory changes in the retina is asignificant factor in the pathogenesis of multiple retinal disordersincluding glaucoma, diabetic retinopathy, and age-related maculardegeneration. Abnormal immune responses arising from physiologicalchanges in microglia, the primary resident innate immune cell in theretina, are thought to drive aspects of disease progression, includingneuronal degeneration and pathological neovascularization (Karlstetteret al., 2015; Silverman and Wong, 2018). Microglia are activated due toa complex interplay between the different cell types of the retina anddiverse pathological pathways. Following activation, microglia cellslose their ramified protrusions, proliferate and rapidly migrate to thedamaged areas and resolve tissue damage. However, sustained presence oftissue stress primes microglia to become over-reactive and results inthe excessive production of pro-inflammatory mediators that favorretinal degenerative changes. A chronic pro-inflammatory environment isa hallmark of retinal degenerative diseases and neurological disordersthat affect vision. Activation of retinal microglia also occurs in amouse model of ischemia/reperfusion injury (I/R), as occurs ininflammatory diseases of the eye, including glaucoma, age relatedmacular degeneration (AMD), diabetic retinopathy and branch veinocclusion. Retinal vascular occlusion, be it by high intra-ocularpressure in the I/R model or thrombus in BVO, causes a decrease in bloodflow within the eye, resulting in retinal ischemia. This causes death ofneurons initiating further activation of microglia.

Enhanced production of pro-inflammatory and angiogenic factors inducesthe formation and growth of new blood vessels from the choroid into thesubretinal space, mimicking features of exudative AMD in a laser-inducedCNV mouse model (Lambert V, et al., Nat. Protoc. 8, 2197-2211 (2013)).Several circumstances, such as ischemia, hypoxia or inflammation, canpromote neovascularization. Pathological ocular angiogenesis,particularly in the retina and choroid, can lead to significant visualimpairment. Diabetic retinopathy, neovascular age-related maculardegeneration (AMD), retinopathy of prematurity, and retinal vesselocclusion are major causes of angiogenesis-related vision loss.

Exudative (wet form) AMD is characterized by serous or hemorrhagicseparation of the retinal pigment epithelium or neurosensory layer.Patients may develop choroidal neovascularization (CNV), which ismanifested as fluid accumulation, hemorrhage, and/or lipid exudation.The earliest stage of diabetic retinopathy (DR) is characterized byretinal vascular abnormalities including microaneurysms (saccularout-pouchings from the capillary wall), intraretinal hemorrhages, andcotton-wool spots (nerve fiber layer infarctions). As the diseaseprogresses, the gradual closure of retinal vessels results in retinalischemia, giving rise to signs including venous abnormalities (beading,loops), intraretinal microvascular abnormalities, and increasing retinalhemorrhage and exudation. Non-proliferative DR is graded as mild,moderate, severe, and very severe according to the presence and extentof the above lesions. The more advanced stage of DR involves theformation of new blood vessels, induced by the retinal ischemia, whichspreads out either from the disc (neovascularization of the disc, NVD)or from elsewhere in the retina (neovascularization elsewhere, NVE). Newvessels extending into the vitreous can cause vitreous hemorrhage, andtractional retinal detachments associated with accompanying contractilefibrous tissue.

To date, the only treatment conclusively demonstrated to be of long-termbenefit for DR is focal laser photocoagulation. The standard treatmentfor patients with AMD is intravitreal injections of anti-VEGF into theeye to slow disease progression, and there have been studies that haveshown that anti-VEGF therapy may be useful in diabetic macular edema(DME). However, there are at present no systemic treatments availablefor ischemic retinopathies or AMD. These would involve less frequentinjections due to retention in microglia and ability to deliverysystemically, avoiding frequent intraocular injections as in currentanti-VEGF therapies.

Therefore, it is an object of the invention to provide compositions andmethods for effective therapies for one or more inflammatory and/orangiogenic diseases of the eye, particularly DME, DR and AMD.

It is another object of the invention to provide compositions andmethods for targeted delivery of one or more active agents to thediseased tissues/cells in the eye via systemic administration withincreased efficacy and reduced side effects.

It is a further object to provide compositions and methods for targeteddelivery of one or more active agents to activated microglia associatedwith one or more inflammatory and/or angiogenic diseases of the eye.

It is also an object to provide compositions and methods effective forinhibiting or reducing pro-inflammatory and/or angiogenic factorsassociated with one or more inflammatory and/or angiogenic diseases ofthe eye.

SUMMARY OF THE INVENTION

Compositions and methods for selective delivery of one or moretherapeutic, prophylactic and/or diagnostic agents to treat and/ordiagnose one or more diseases and/or disorders of the eye have beendeveloped. The compositions deliver one or more therapeutic,prophylactic and/or diagnostic agents selectively to activatedmicroglial cells to treat and/or diagnose diseases tissues/cells of theeye.

Compositions include hydroxyl-terminated dendrimer complexed, covalentlyconjugated or intra-molecularly dispersed or encapsulated with one ormore receptor tyrosine kinase inhibitors in an amount effective toreduce the number or activity of the activated microglia and macrophagesin the retina and/or the choroid in a subject in need thereof. In someembodiments, the receptor tyrosine kinase inhibitor is an inhibitor ofvascular endothelial growth factor receptors such as sunitinib,sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib,dasatinib, nintedanib, motesanib, and analogues thereof. Preferably, thereceptor tyrosine kinase inhibitor is sunitinib or an analogue thereof.In some embodiments, the diagnostic agents are dyes, such as fluorescentdyes, Near infra-red dyes, SPECT imaging agents, PET imaging agents andradioisotopes. Preferably, the diagnostic agent is the fluorescent dyeindocyanine green (ICG).

In some embodiments, the dendrimer is a generation 4, generation 5,generation 6, generation 7, generation 8, generation 9, or generation 10PAMAM dendrimer. In some embodiments, the one or more therapeutic,prophylactic and/or diagnostic agents are covalently conjugated to thedendrimers.

In some embodiments, the one or more therapeutic, prophylactic and/ordiagnostic agents are at a concentration by weight of agent to dendrimerconjugate of between about 0.01% weight to weight (w/w) to about 30%w/w, about 1% w/w to about 25% w/w, about 5% w/w to about 20% w/w, andabout 10% w/w to about 15% w/w.

In some embodiments, one or more spacers or linkers between a dendrimerand an agent are added to provide a releasable (or cleavable) ornon-releasable (or non-cleavable) form of the dendrimer-agent complexesin vivo. In some embodiments, the attachment occurs via an appropriatespacer that provides an ester bond between the agent and the dendrimer.In some embodiments, the attachment occurs via an appropriate spacerthat provides an ether bond between the agent and the dendrimer. In someembodiments, the attachment occurs via an appropriate spacer thatprovides an amide bond between the agent and the dendrimer. In preferredembodiments, one or more spacers/linkers between a dendrimer and anagent are tailored to achieve desired and effective release kinetics invivo.

The compositions are suitable for treating and/or diagnosing one or moreinflammatory and/or angiogenic diseases of the eye, for example,age-related macular degeneration (AMD), retinitis pigmentosa, opticneuritis, uveitis, retinal detachment, temporal arteritis, retinalischemia, arteriosclerotic retinopathy, hypertensive retinopathy,retinal artery blockage, retinal vein blockage, diabetic retinopathy,macular edema, retinal neovascularization, and choroidalneovascularization.

Methods of making the dendrimer compositions are provided. Dosage formsand pharmaceutical formulations including an effective amount of thedendrimer compositions for administration to a subject in need thereofare also provided.

Methods of treating and/or diagnosing one or more diseases and/ordisorders of the eye by administering to a subject in need thereof aneffective amount of the compositions are described. The methods areeffective in treating and/or diagnosing one or more diseases and/ordisorders of the eye, including age-related macular degeneration (AMD),retinitis pigmentosa, optic neuritis, uveitis, retinal detachment,temporal arteritis, retinal ischemia, arteriosclerotic retinopathy,hypertensive retinopathy, retinal artery blockage, retinal veinblockage, diabetic retinopathy, macular edema, retinalneovascularization, and choroidal neovascularization. In particular, themethods are effective for treating and/or diagnosing one or morediseases and/or disorders of the eye associated with activated microgliawithin the eye and surrounding tissue. Typically, the compositions areadministered in an amount effective to target the activated microglia,retinal pigment epithelia (RPE) cells, and/or choroidal neovascular(CNV) lesions, and/or to alleviate one or more symptoms of the one ormore one or more diseases and/or disorders of the eye.

Methods of administering the compositions and pharmaceuticalformulations are also provided. Typically, the compositions andpharmaceutical formulations are administered via one or more systemicroutes daily, weekly, biweekly, monthly, bimonthly, or less frequently.In some embodiments, the compositions and pharmaceutical formulationsare administered via one or more systemic routes once every four weeksor less frequently. In preferred embodiments, the compositions andpharmaceutical formulations are administered via the intravenous,subcutaneous, or oral route.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schemes showing chemical reaction steps for thesynthesis of a dendrimer-sunitinib conjugate. Sunitinib is conjugated tothe dendrimer via a hydroxymethyl linkage (FIG. 1A), and an amidelinkage (FIG. 1B), respectively.

FIGS. 2A and 2B are bar graphs showing levels of isolectin (FIG. 2A),and IBA-1 (FIG. 2B), showing signal area (μm²) for each of 4 hours (4 h)and 24 hours (24 h), at day 1, 3, 7 and 14 post-laser, respectively, asanalyzed by optical coherence tomography with ICG imaging at 4 or 24 hrpost-dose with D-ICG. FIG. 2C is a bar graph showing corrected totallesion fluorescence over a period of 28 days after a single systemicdendrimer-indocyanine green (D-ICG) dose given 24 hr after laser injurylocalized to the choroidal neovascularization (CNV) lesion in C57BL/6mice.

FIG. 3 is a bar graph showing mean area of choroidal neovascularization(CNV) (mm²) in the eyes of mice treated with vehicle, aflibercept, acleavable sunitinib analog (D-CSA) at a low dose (D-CSA low) or a highdose (D-CSA high), a non-cleavable sunitinib analog (D-NSA) at a lowdose (D-NSA low) or a high dose (D-NSA high), and free sunitinibadministered 24 hr after laser-induced rupture of Bruch's membrane inthe eyes of C57BL/6 mice (n=8/group for all except D-NSA High wheren=6). P values are indicated as compared to the vehicle control.

FIG. 4A is a bar graph showing mean area of choroidal neovascularization(CNV) (mm²) in the eyes of mice treated with free sunitinib, a cleavablesunitinib analog (D-CSA), a non-cleavable sunitinib analog (D-NSA), andaflibercept administered 24 hr after laser-induced rupture of Bruch'smembrane in the eyes of C57BL/6 mice (n=8/group) at day 7 and day 14post-treatment, respectively. FIG. 4B is a line plot showing plasmaconcentration (μg/ml) of dendrimer sunitinib analog conjugates over timefor a period of 0-72 hours.

FIG. 5 is a reaction scheme showing one synthesis strategy of N,N-didesethyl sunitinib azide with an amide linkage.

FIGS. 6A and 6B are schemes showing chemical reaction steps for thesynthesis of an exemplary dendrimer-sunitinib conjugate via firstsynthesizing dendrimer-hexynoic-acid conjugate (FIG. 6A), prior to thestep of synthesizing dendrimer-didesethyl-sunitinib amide-conjugate(FIG. 6B). G4 PAMAM dendrimer is used as an exemplary dendrimer.

FIG. 7 is a line graph showing in vitro release profile (loss of linkerwith AVT-4517% w/w) of D-didesethyl sunitinib conjugate (D-4517) pH 7.4and pH 5.5 over a period of 15 days with esterases at 37° C. mimickingplasma and intracellular conditions respectively.

FIG. 8 is a line graph showing plasma concentration in μg/mL over timein Female C57/B16 mice injected IP with 5 or 50 mg/kg D-4517.

FIGS. 9A and 9B are line graphs showing plasma concentration in μg/mLover time (0-24 hours) for male and female groups of Sprague-Dawley ratsthat received daily IP injections of 12 mg/kg D-4517 and daily oral doseof 30 mg/kg sunitinib (40.21 mg/kg of sunitinib malate) on Day 1 (FIG.9A), and Day 14 (FIG. 9B), respectively.

FIG. 10 is a bar graph showing mean area of choroidal neovascularization(CNV) (μm²) in the eyes of mice treated with vehicle, aflibercept (40μg), and three dose levels of D-didesethyl sunitinib conjugate (D-4517)at 2, 10 and 50 mg/kg in a single subcutaneous dose administered 24 hrafter laser-induced rupture of Bruch's membrane in the eyes of C57BL/6mice at day 14 post-treatment.

FIG. 11 is a scheme showing the synthesis of an exemplarydendrimer-conjugate (D-4517.2) in which N, N-didesethyl sunitinib isconjugated to a dendrimer with ether linkages for enhanced in vivostability.

FIG. 12 is a schematic showing the chemical structure of compoundD-4517.2.

FIG. 13 is a bar graph showing drug release percentage by weight(0.0%-0.50%) of D-didesethyl sunitinib conjugate, D-4517.2, in human,mouse, and rat plasma conditions over time points for each of 4, 24 and48 hours, respectively.

FIG. 14 is a synthesis scheme for Dendrimer-N-Acetyl-L-cysteine methylester conjugate (Dendrimer-NAC-carboxymethylated conjugate).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms “active agent” or “biologically active agent” are therapeutic,prophylactic or diagnostic agents used interchangeably to refer to achemical or biological compound that induces a desired pharmacologicaland/or physiological effect, which may be prophylactic, therapeutic ordiagnostic. These may be a nucleic acid, a nucleic acid analog, a smallmolecule having a molecular weight less than 2 kDa, more typically lessthan 1 kDa, a peptidomimetic, a protein or peptide, carbohydrate orsugar, lipid, or surfactant, or a combination thereof. The terms alsoencompass pharmaceutically acceptable, pharmacologically activederivatives of active agents, including, but not limited to, salts,esters, amides, prodrugs, active metabolites, and analogs.

The term “pharmaceutically acceptable salts” is art-recognized, andincludes relatively non-toxic, inorganic and organic acid addition saltsof compounds. Examples of pharmaceutically acceptable salts includethose derived from mineral acids, such as hydrochloric acid and sulfuricacid, and those derived from organic acids, such as ethanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid. Examples of suitableinorganic bases for the formation of salts include the hydroxides,carbonates, and bicarbonates of ammonia, sodium, lithium, potassium,calcium, magnesium, aluminum, and zinc. Salts may also be formed withsuitable organic bases, including those that are non-toxic and strongenough to form such salts. For purposes of illustration, the class ofsuch organic bases may include mono-, di-, and trialkylamines, such asmethylamine, dimethylamine, and triethylamine; mono-, di- ortrihydroxyalkylamines such as mono-, di-, and triethanolamine; aminoacids, such as arginine and lysine; guanidine; N-methylglucosamine;N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine;ethylenediamine; N-benzylphenethylamine;

The term “therapeutic agent” refers to an active agent that can beadministered to treat one or more symptoms of a disease or disorder.

The term “diagnostic agent” refers to an active agent that can beadministered to reveal, pinpoint, and define the localization of apathological process. The diagnostic agents can label target cells thatallow subsequent detection or imaging of these labeled target cells. Insome embodiments, diagnostic agents can, via dendrimer or suitabledelivery vehicles, target/bind activated microglia, activatedmacrophages, and/or RPE cells.

The term “prophylactic agent” refers to an active agent that can beadministered to prevent disease or to prevent certain conditions.

The phrase “pharmaceutically acceptable”, or “biocompatible” refers tocompositions, polymers and other materials and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. Thephrase “pharmaceutically acceptable carrier” refers to pharmaceuticallyacceptable materials, compositions or vehicles, such as a liquid orsolid filler, diluent, solvent or encapsulating material involved incarrying or transporting any subject composition, from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of a subject composition and not injurious to thepatient.

The term “therapeutically effective amount” refers to an amount of thetherapeutic agent that, when incorporated into and/or onto dendrimers,produces some desired effect at a reasonable benefit/risk ratioapplicable to any medical treatment. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation. In someembodiments, the term “effective amount” refers to an amount of atherapeutic agent or prophylactic agent to reduce or diminish thesymptoms of one or more eye diseases or disorders, such as reducinginflammation by reducing or inhibiting one or more pro-inflammatorycytokines and/cells associated with the diseased tissues/cells in theeye. In the case of retinal and/or choroidal neovascularization, aneffective amount of the drug may have the effect in reducing retinaland/or choroidal angiogenesis; inhibiting to some extent vascularendothelial cell growth/proliferation; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations.

The terms “inhibit” or “reduce” in the context of inhibition, mean toreduce or decrease in activity and quantity. This can be a completeinhibition or reduction in activity or quantity, or a partial inhibitionor reduction. Inhibition or reduction can be compared to a control or toa standard level. Inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95,99, or 100%. For example, dendrimer compositions including one or moretherapeutic agents may inhibit or reduce the activity and/or quantity ofactivated microglia and macrophages in the diseased retina and/orchoroid of a subject by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%,95%, or 99% from the activity and/or quantity of the same cells inequivalent diseased tissues of subjects that did not receive, or werenot treated with the dendrimer compositions (i.e., un-conjugated activeagents). In some embodiments, the inhibition and reduction are comparedat mRNAs, proteins, cells, tissues and organs levels. For example, aninhibition and/or reduction in pro-inflammatory cytokines (e.g., TNF-α,interleukin-1β (IL-1β), or interferon-γ (IFN-γ)) secreted by theactivated microglia and macrophages in the diseased retina and/orchoroid.

The term “treating” or “preventing” a disease, disorder or conditionfrom occurring in an animal which may be predisposed to the disease,disorder and/or condition but has not yet been diagnosed as having it;inhibiting the disease, disorder or condition, e.g., impeding itsprogress; and relieving the disease, disorder, or condition, e.g.,causing regression of the disease, disorder and/or condition. Treatingthe disease or condition includes ameliorating at least one symptom ofthe particular disease or condition, even if the underlyingpathophysiology is not affected, such as treating the pain of a subjectby administration of an analgesic agent even though such agent does nottreat the cause of the pain. Desirable effects of treatment includedecreasing the rate of disease progression, ameliorating or palliatingthe disease state, and remission or improved prognosis. For example, anindividual is successfully “treated” if one or more symptoms associatedwith an eye disease or disorder are mitigated or eliminated, including,but are not limited to, reducing the proliferation of pro-inflammatorycells, decreasing symptoms resulting from the disease, enhancing orrestoring vision, decreasing the extent and rate of vision loss,increasing the quality of life of those suffering from the disease,decreasing the dose of other medications required to treat the disease,delaying the progression of the disease, and/or prolonging survival ofindividuals.

The term “biodegradable” refers to a material that will degrade or erodeunder physiologic conditions to smaller units or chemical species thatare capable of being metabolized, eliminated, or excreted by thesubject. The degradation time is a function of composition andmorphology.

The term “dendrimer” includes, but is not limited to, a moleculararchitecture with an interior core, interior layers (or “generations”)of repeating units regularly attached to this initiator core, and anexterior surface of terminal groups attached to the outermostgeneration.

The term “functionalize” means to modify a compound or molecule in amanner that results in the attachment of a functional group or moiety.For example, a molecule may be functionalized by the introduction of amolecule which makes the molecule a strong nucleophile or strongelectrophile.

The term “targeting moiety” refers to a moiety that localizes to or awayfrom a specific locale. The moiety may be, for example, a protein,nucleic acid, nucleic acid analog, carbohydrate, or small molecule. Theentity may be, for example, a therapeutic compound such as a smallmolecule, or a diagnostic entity such as a detectable label. The localemay be a tissue, a particular cell type or cell activation state, or asubcellular compartment. In some embodiments, the targeting moietydirects the localization of an active agent.

The term “prolonged residence time” refers to an increase in the timerequired for an agent to be cleared from a patient's body, or organ ortissue of that patient. In certain embodiments, “prolonged residencetime” refers to an agent that is cleared with a half-life that is 10%,20%, 50% or 75% longer than a standard of comparison such as acomparable agent without conjugation to a delivery vehicle such as adendrimer. In certain embodiments, “prolonged residence time” refers toan agent that is cleared with a half-life of 2, 5, 10, 20, 50, 100, 200,500, 1000, 2000, 5000, or 10000 times longer than a standard ofcomparison such as a comparable agent without a dendrimer thatspecifically target specific cell types associated with tumors.

The terms “incorporated” and “encapsulated” refer to incorporating,formulating, or otherwise including an active agent into and/or onto acomposition that allows for release, such as sustained release, of suchagent in the desired application. The active agent or other material canbe incorporated into a dendrimer, including to one or more surfacefunctional groups of such dendrimer (by covalent, ionic, or otherbinding interaction), physical admixture, enveloping the agent withinthe dendritic structure, encapsulated inside the dendritic structure,etc.

II. Compositions

Dendrimer complexes suitable for delivering one or more active agent,particularly one or more active agents to prevent, treat or diagnose oneor more diseases or disorders of the eye.

Compositions of dendrimer complexes include one or more prophylactic,therapeutic, and/or diagnostic agents encapsulated, associated, and/orconjugated in the dendrimer complex at a concentration by weight ofabout 0.01% weight to weight (w/w) to about 30% w/w, about 1% w/w toabout 25% w/w, about 5% w/w to about 20% w/w, and about 10% w/w to about15% w/w. In some embodiments, prophylactic, therapeutic, and/ordiagnostic agents are covalently conjugated to the dendrimer via one ormore linkages selected from disulfide, ester, ether, thioester,carbamate, carbonate, hydrazine, and amide, optionally via one or morespacers. Preferably, hydroxyl groups of hydroxyl-terminated dendrimersare covalently conjugated to one or more active agents via at least oneether linkage, optionally via one or more linkers/spacers. In preferredembodiments, surface groups of hydroxyl-terminated dendrimers aremodified via etherification reaction prior to conjugation to one or morelinkers and the active agent. Where one or more linkers are presentbetween dendrimers and active agents, the covalent bond between thesurface groups of dendrimers and the linkers are ether bonds. In otherembodiments, at dendrimer generation 3.5, alkyne functional groups areintroduced using a polyethyl glycol (PEG) linker with an amine at oneend and a hexyne at the other end to produce a generation 4 bifunctionaldendrimer. An exemplary bifunctional dendrimer is shown as compound 1 inFIG. 11 with 7 alkyne arms and 57 hydroxyl groups on the surface.

In some embodiments, the spacer is a prophylactic, therapeutic, and/ordiagnostic agent, such as sunitinib. Exemplary active agents includeantiangiogenic agents, anti-inflammatory drugs, and anti-infectiveagents.

The presence of the additional agents can affect the zeta-potential, orthe surface charge of the particle. In one embodiment, the zetapotential of the dendrimers is −100 mV and 100 mV, between −50 mV and 50mV, between −25 mV and 25 mV, between −20 mV and 20 mV, between −10 mVand 10 mV, between −10 mV and 5 mV, between −5 mV and 5 mV, or between−2 mV and 2 mV. In a preferred embodiment, the surface charge is neutralor near-neutral. The range above is inclusive of all values from −100 mVto 100 mV.

A. Dendrimers

Dendrimers are three-dimensional, hyperbranched, monodispersed, globularand polyvalent macromolecules having a high density of surface endgroups (Tomalia, D. A., et al., Biochemical Society Transactions, 35, 61(2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)). Dueto their unique structural and physical features, dendrimers are usefulas nano-carriers for various biomedical applications including targeteddrug/gene delivery, imaging and diagnosis (Sharma, A., et al., RSCAdvances, 4, 19242 (2014); Caminade, A.-M., et al., Journal of MaterialsChemistry B, 2, 4055 (2014); Esfand, R., et al., Drug Discovery Today,6, 427 (2001); and Kannan, R. M., et al., Journal of Internal Medicine,276, 579 (2014)).

Dendrimer surface groups have a significant impact on theirbiodistribution (Nance, E., et al., Biomaterials, 101, 96 (2016)).Hydroxyl terminated generation 4 PAMAM dendrimers (˜4 nm size) withoutany targeting ligand cross the impaired BBB upon systemic administrationin a rabbit model of cerebral palsy (CP) significantly more (>20 fold)as compared to healthy controls, and selectively target activatedmicroglia and astrocytes (Lesniak, W. G., et al., Mol Pharm, 10 (2013)).

The term “dendrimer” includes a molecular architecture with an interiorcore and layers (or “generations”) of repeating units which are attachedto and extend from this interior core, each layer having one or morebranching points, and an exterior surface of terminal groups attached tothe outermost generation. In some embodiments, dendrimers have regulardendrimeric or “starburst” molecular structures.

Generally, dendrimers have a diameter from about 1 nm up to about 50 nm,more preferably from about 1 nm to about 20 nm, from about 1 nm to about10 nm, or from about 1 nm to about 5 nm. In some embodiments, thediameter is between about 1 nm to about 2 nm. Conjugates are generallyin the same size range, although large proteins such as antibodies mayincrease the size by 5-15 nm. In general, agent is encapsulated in aratio of agent to dendrimer of between 1:1 to 4:1 for the largergeneration dendrimers. In preferred embodiments, the dendrimers have adiameter effective to penetrate ocular tissue and to be retained intarget cells for a prolonged period of time.

In some embodiments, dendrimers have a molecular weight between about500 Daltons to about 100,000 Daltons, preferably between about 500Daltons to about 50,000 Daltons, most preferably between about 1,000Daltons to about 20,000 Dalton.

Suitable dendrimers scaffolds that can be used include poly(amidoamine),also known as PAMAM, or STARBURST™ dendrimers; polypropylamine (POPAM),polyethylenimine, polylysine, polyester, iptycene, aliphaticpoly(ether), and/or aromatic polyether dendrimers. The dendrimers canhave carboxylic, amine and/or hydroxyl terminations. In preferredembodiments, the dendrimers have hydroxyl terminations. Each dendrimerof the dendrimer complex may be same or of similar or different chemicalnature than the other dendrimers (e.g., the first dendrimer may includea PAMAM dendrimer, while the second dendrimer may be a POPAM dendrimer).

The term “PAMAM dendrimer” means poly(amidoamine) dendrimer, which maycontain different cores, with amidoamine building blocks, and can havecarboxylic, amine and hydroxyl terminations of any generation including,but not limited to, generation 1 PAMAM dendrimers, generation 2 PAMAMdendrimers, generation 3 PAMAM dendrimers, generation 4 PAMAMdendrimers, generation 5 PAMAM dendrimers, generation 6 PAMAMdendrimers, generation 7 PAMAM dendrimers, generation 8 PAMAMdendrimers, generation 9 PAMAM dendrimers, or generation 10 PAMAMdendrimers. In the preferred embodiment, the dendrimers are soluble inthe formulation and are generation (“G”) 4, 5 or 6 dendrimers. Inpreferred embodiments, dendrimers have a plurality of hydroxyl groupsattached to their functional surface groups.

Methods for making dendrimers are known to those of skill in the art andgenerally involves a two-step iterative reaction sequence that producesconcentric shells (generations) of dendritic β-alanine units around acentral initiator core (e.g., ethylenediamine-cores). Each subsequentgrowth step represents a new “generation” of polymer with a largermolecular diameter, twice the number of reactive surface sites, andapproximately double the molecular weight of the preceding generation.Dendrimer scaffolds suitable for use are commercially available in avariety of generations. Preferable, the dendrimeric compounds are basedon generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 dendrimeric scaffolds.Such scaffolds have, respectively, 4, 8, 16, 32, 64, 128, 256, 512,1024, 2048, and 4096 reactive sites. Thus, the dendrimeric compoundsbased on these scaffolds have the corresponding number of combinedtargeting moieties and modulators.

In some embodiments, the dendrimers include a plurality of hydroxylgroups. Some exemplary high-density hydroxyl groups-containingdendrimers include commercially available polyester dendritic polymersuch as hyperbranched 2,2-Bis(hydroxyl-methyl)propionic acid polyesterpolymer (for example, hyperbranched bis-MPA polyester-64-hydroxyl,generation 4), dendritic polyglycerols.

In some embodiments, the high-density hydroxyl groups-containingdendrimers are oligo ethylene glycol (OEG)-like dendrimers. For example,a generation 2 OEG dendrimer (D2-OH-60) can be synthesized using highlyefficient, robust and atom economical chemical reactions such as Cu (I)catalyzed alkyne-azide click and photo catalyzed thiol-ene clickchemistry. Highly dense polyol dendrimer at very low generation inminimum reaction steps can be achieved by using an orthogonalhypermonomer and hypercore strategy, for example as described in WO2019094952. In some embodiments, dendrimer backbone has non-cleavablepolyether bonds throughout the structure to avoid the disintegration ofdendrimer in vivo, and to allow the elimination of such dendrimers as asingle entity from the body (non-biodegradable).

In some embodiments, the dendrimer is able to specifically target aparticular tissue region and/or cell type, preferably activatedmicroglia and macrophages associated with one or more eye diseases. Inpreferred embodiments, the dendrimer is able to specifically target aparticular tissue region and/or cell type without addition of atargeting moiety.

In preferred embodiments, the dendrimers have a plurality of hydroxyl(—OH) groups on the surface of the dendrimers. The preferred surfacedensity of hydroxyl (—OH) groups is at least 1 OH group/nm² (number ofhydroxyl surface groups/surface area in nm²). For example, in someembodiments, the surface density of hydroxyl groups is more than 2, 3,4, 5, 6, 7, 8, 9, 10; preferably at least 10, 15, 20, 25, 30, 35, 40,45, 50, or more than 50 surface groups/surface area in nm². In furtherembodiments, the surface density of hydroxyl (—OH) groups is betweenabout 1 and about 50, preferably 5-20 OH group/nm² (number of hydroxylsurface groups/surface area in nm²) while having a molecular weight ofbetween about 500 Da and about 10 kDa. In preferred embodiments, thepercentage of free, i.e., un-conjugated hydroxyl groups out of totalsurface groups (conjugated and un-conjugated) on the dendrimer is morethan 70%, 75%, 80%, 85%, 90%, 95%, and/or less than 100%. In the case ofgeneration 4 PAMAM dendrimers, the preferred number of free, i.e.,un-conjugated hydroxyl groups is more than 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, or 63 out of total 64 surface terminals/groups.In further embodiments, the hydroxyl terminated dendrimers have aneffective number of free hydroxyl groups for selective targeting toactivated microglia, activated microphages, and/or RPE cells associatedwith one or more diseases and/or disorders of the eye.

In some embodiments, the dendrimers may have a fraction of the hydroxylgroups exposed on the outer surface, with the others in the interiorcore of the dendrimers. In preferred embodiments, the dendrimers have avolumetric density of hydroxyl (—OH) groups of at least 1 OH group/nm³(number of hydroxyl groups/volume in nm³). For example, in someembodiments, the volumetric density of hydroxyl groups is 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10, 15, 20, 25, 30, 35, 40, 45, and 50hydroxyl groups/volume in nm³. In some embodiments, the volumetricdensity of hydroxyl groups is between about 4 to about 50 hydroxylgroups/nm³, preferably between about 5 to about 30 hydroxyl groups/nm³,more preferably between about 10 to about 20 hydroxyl groups/nm³.

B. Coupling Agents and Spacers

Dendrimer complexes can be formed of therapeutically active agents orcompounds conjugated or attached to a dendrimer, a dendritic polymer ora hyperbranched polymer. Optionally, the active agents are conjugated tothe dendrimers via one or more spacers/linkers via different linkagessuch as disulfide, ester, carbonate, carbamate, thioester, hydrazine,hydrazides, and amide linkages. The one or more spacers/linkers betweena dendrimer and an agent can be designed to provide a releasable (orcleavable) or non-releasable (or non-cleavable) form of thedendrimer-active complexes in vivo. In some embodiments, the attachmentoccurs via an appropriate spacer that provides an ester bond between theagent and the dendrimer. In some embodiments, the attachment occurs viaan appropriate spacer that provides an amide bond between the agent andthe dendrimer. In preferred embodiments, one or more spacers/linkersbetween a dendrimer and an agent are added to achieve a desired andeffective release kinetics in vivo. In further embodiments, theconjugation of dendrimer and/or linker does not significantly affect theactivities of the active agents. For example, in the case of VEGFR TKRinhibitors, they retain their binding affinity towards one or more ofVEGFR TKR after conjugation to dendrimers at a level comparable tounconjugated VEGFR TKR inhibitors.

The term “spacer” includes moieties and compositions used for linking atherapeutically active agent to the dendrimer. The spacer can be eithera single chemical entity or two or more chemical entities linkedtogether to bridge the dendrimer and the active agent. The spacers caninclude any small chemical entity, peptide or polymers havingsulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone, andcarbonate terminations.

The spacer can be chosen from among a class of compounds terminating insulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone andcarbonate group. The spacer can include thiopyridine terminatedcompounds such as dithiodipyridine, N-Succinimidyl3-(2-pyridyldithio)-propionate (SPDP), Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or Sulfo-LC-SPDP.The spacer can also include peptides wherein the peptides are linear orcyclic essentially having sulfhydryl groups such as glutathione,homocysteine, cysteine and its derivatives, arg-gly-asp-cys (RGDC),cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-D-Tyr-Cys),and cyclo(Arg-Ala-Asp-d-Tyr-Cys). In some embodiments, the spacerincludes a mercapto acid derivative such as 3 mercapto propionic acid,mercapto acetic acid, 4 mercapto butyric acid, thiolan-2-one, 6mercaptohexanoic acid, 5 mercapto valeric acid and other mercaptoderivatives such as 2 mercaptoethanol and 2 mercaptoethylamine. In someembodiments, the spacer includes thiosalicylic acid and its derivatives,(4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene,(3-[2-pyridithio]propionyl hydrazide. In some embodiments, the spacerincludes maleimide terminations wherein the spacer includes polymer orsmall chemical entity such as bis-maleimido diethylene glycol andbis-maleimido triethylene glycol, Bis-Maleimidoethane, andbismaleimidohexane. In some embodiments, the spacer includesvinylsulfone such as 1,6-Hexane-bis-vinylsulfone. In some embodiments,the spacer includes thioglycosides such as thioglucose. In otherembodiments, the spacer includes reduced proteins such as bovine serumalbumin and human serum albumin, any thiol terminated compound capableof forming disulfide bonds. In particular embodiments, the spacerincludes polyethylene glycol having maleimide, succinimidyl and thiolterminations.

The therapeutically active agent, imaging agent, and/or targeting moietycan be either covalently attached or intra-molecularly dispersed orencapsulated. The dendrimer is preferably a PAMAM dendrimer ofgeneration 1 (G1), G2, G3, G4, G5, G6, G7, G8, G9 or G10, havingcarboxylic, hydroxyl, or amine terminations. In preferred embodiments,the dendrimer is linked to active agents via a spacer ending in ether oramide bonds.

In some embodiments, a non-releasable form of the dendrimer/active agentcomplex provides enhanced therapeutic efficacy as compared to areleasable or cleavable form of the same dendrimer/active agent complex.Therefore, in some embodiments, one or more active agent(s) isconjugated to the dendrimer via a spacer that is attached to thedendrimer in a non-releasable manner, for example, by an ether or amidebond. In some embodiments, one or more active agent(s) is attached tothe spacer in a non-releasable manner, for example, by an ether or amidebond. Therefore, in some embodiments, one or more active agent(s) isattached to the dendrimer via a spacer that is attached to thedendrimer, and to the active agent(s) in a non-releasable manner. In anexemplary embodiment, one or more active agent(s) is attached to thedendrimer via a spacer that is attached to the dendrimer and the activeagent(s) via amide and/or ether bonds. An exemplary spacer ispolyethylene glycol (PEG).

1. Dendrimer Conjugation to Active Agents Via Ether Linkages

In some embodiments, the compositions include a hydroxyl-terminateddendrimer conjugated to an active agent via an ether linkage, optionallywith one or more linkers/spacers.

In preferred embodiments, the covalent bonds between the surface groupsof the dendrimers and the linkers, or the dendrimers and the activeagent (if conjugated without any linking moieties) are stable under invivo conditions, i.e., minimally cleavable when administered to asubject and/or excreted intact from the body. For example, in preferredembodiments, less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%,0.1%, or less than 0.1% of the total dendrimer complexes have activeagent cleaved within 24 hours, or 48 hours, or 72 hours after in vivoadministration. In one embodiment, the covalent bonds are ether bonds.In further preferred embodiments, the covalent bond between the surfacegroups of the dendrimers and the linkers, or the dendrimers and theactive agent (if conjugated without any linking moieties), are nothydrolytically or enzymatically cleavable bonds, such as ester bonds.

In some embodiments, one or more hydroxyl groups of hydroxyl-terminateddendrimers conjugate to one or more linking moieties and one or moreactive agents via one or more ether bonds as shown in Formula (I) below.

wherein D is a G2 to G10 poly(amidoamine) (PAMAM) dendrimer; L is one ormore linking moieties or spacers; X is an active agent or analogthereof; n is an integer from 1 to 100; and m is an integer from 16 to4096; and

Y is a linker selected from secondary amides (—CONH—), tertiary amides(—CONR—), sulfonamide (—S(O)₂—NR—), secondary carbamates (—OCONH—;—NHCOO—), tertiary carbamates (—OCONR—; —NRCOO—), carbonate(—O—C(O)—O—), ureas (—NHCONH—; —NRCONH—; —NHCONR—, —NRCONR—), carbinols(—CHOH—, —CROH—), disulfide groups, hydrazones, hydrazides, and ethers(—O—), wherein R is an alkyl group, an aryl group, or a heterocyclicgroup. Preferably, Y is a bond or linkage that is minimally cleavable invivo.

In some embodiments, X is an inhibitor of vascular endothelial growthfactor receptor (VEGFR) and/or TIE2 receptor tyrosine kinases.

In a preferred embodiment, Y is a secondary amide (—CONH—).

In one embodiment, D is a generation 4 PAMAM dendrimer; L is one or morelinking or spacer moieties; X is sunitinib, or analog thereof; n isabout 5-15; m is an integer about 49-59; and where n+m=64.

In another embodiment, D is a generation 4 PAMAM dendrimer; L is one ormore linking or spacer moieties; X is N, N-didesethyl sunitinib; Y is asecondary amide (—CONH—); n is about 5-15; m is an integer about 49-59;and where n+m=64.

In a specific embodiment, the Formula I has the following structure(also referred to as D-4517.2):

Structure I: Chemical Structure of D-4517.2

C. Therapeutic, Prophylactic and Diagnostic Agents

Dendrimers have the advantage that multiple therapeutic, prophylactic,and/or diagnostic agents can be delivered with the same dendrimers. Insome embodiments, one or more types of active agents are encapsulated,complexed or conjugated to the dendrimer. In another embodiment, thedendrimers are covalently linked to at least one detectable moiety, inan amount effective to detect one or more diseased or injuredcells/tissues in the subject. In particular embodiments, the dendrimercomposition has multiple agents, such as an immunotherapeutic agent, ananti-seizure agent, a steroid to decrease swelling, an antibiotic, ananti-angiogenic agent, and/or a diagnostic agent, complexed with orconjugated to the dendrimers. In some embodiment, the dendrimers arecomplexed with or conjugated to two or more different classes of activeagents, providing simultaneous delivery with different or independentrelease kinetics at the target site. For example, both sunitinib and ananti-inflammatory agent can be conjugated onto the same dendrimer fordelivery to target cells/tissues. In a further embodiment, dendrimercomplexes each carrying different classes of active agents areadministered simultaneously for a combination treatment. In someembodiments, one or more therapeutic agents targeting the underlyingcause of the disease or condition, and one or more therapeutic agentsrelieving one or more symptoms of the disease or condition.

Suitable active agents include therapeutic, diagnostic, and/orprophylactic agents. The agent can be a biomolecule, such as an enzyme,protein, polypeptide, or nucleic acid or a small molecule agent (e.g.,molecular weight less than 2000 Dalton, preferably less than 1500Dalton, more preferably 300-700 Dalton), including organic, inorganic,and organometallic agents. The agent can be encapsulated within thedendrimers, dispersed within the dendrimers, and/or associated with thesurface of the dendrimer, either covalently or non-covalently. Exemplarytherapeutic agents include anti-inflammatory drugs, anti-angiogenicagents, anti-oxidants, vasodilators, neuroactive agents, neuroprotectiveagents and anti-infective agents. In some embodiments, the dendrimer islinked to the targeting moiety, imaging agents, and/or therapeuticagents via a linker or spacer ending in disulfide, ester, ether,thioester, carbamate, carbonate, hydrazine, or amide bonds.

The dendrimers can be used to deliver one or more additional activeagents, particularly one or more active agents to prevent or treat oneor more symptoms of the eye diseases. Exemplary therapeutic agentsadministered with dendrimers include tyrosine kinase inhibitors such asVEGFR tyrosine kinase inhibitors. In a preferred embodiment, the agentsare small molecule tyrosine kinase inhibitors.

Representative anti-angiogenesis agents include, but are not limited to,antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and otheranti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanimsodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigmentepithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib(CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12(IL-12); thalidomide (THALOMID®) and derivatives thereof such aslenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozymeinhibitors such as ANGIOZYME® (Sirna Therapeutics); multifunctionalantiangiogenic agents such as NEOVASTAT® (AE-941) (Aeterna Laboratories,Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such assunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grownfactor receptor such as panitumumab (VECTIBIX®) and cetuximab(ERBITUX®), as well as other anti-angiogenesis agents known in the art.

Other active agents suitable for anti-angiogenic therapies include thosetargeting members of the platelet-derived growth factor family,epidermal growth factor family, fibroblast growth factor family,transforming growth factor-β superfamily (TGF-β1, activins, follistatinand bone morphogenetic proteins), angiopoietin-like family, galectinsfamily, integrin superfamily, as well as pigment epithelium derivedfactor, hepatocyte growth factor, angiopoietins, endothelins,hypoxia-inducible factors, insulin-like growth factors, cytokines,matrix metalloproteinases and their inhibitors and glycosylationproteins.

Tyrosine Kinase Inhibitor

In some embodiments, the dendrimers are complexed or conjugated with oneor more tyrosine kinase inhibitors.

Tyrosine kinases are important cellular signaling proteins that have avariety of biological activities including cell proliferation andmigration. Multiple kinases are involved in angiogenesis, includingreceptor tyrosine kinases such as the vascular endothelial growth factorreceptor (VEGFR). Anti-angiogenic tyrosine kinase inhibitors in clinicaldevelopment primarily target VEGFR-1, -2, -3, epidermal growth factorreceptor (EGFR), platelet-derived growth factor receptor (PDGFR),PDGFR-β, KIT, fms-related tyrosine kinase 3 (FLT3), colony stimulatingfactor-1 receptor (CSF-1R), Raf, and RET.

VEGFR Inhibitor

In some embodiments, the dendrimers are complexed or conjugated with oneor more VEGFR tyrosine kinase inhibitors. The VEGFR family includesthree related receptor tyrosine kinases, known as VEGFR-1, -2, and -3,which mediate the angiogenic effect of VEGF ligands (Hicklin D J, EllisL M. J Clin Oncol. (2005), 23(5):1011-27). The VEGF family encoded inthe mammalian genome includes five members: VEGF-A, VEGF-B, VEGF-C,VEGF-D, and placental growth factor (P1GF). VEGFs are importantstimulators of proliferation and migration of endothelial cells.Increased expression of the angiogenic factor VEGF-A promotes threecommon aging-related eye conditions—“wet” and “dry” forms of age-relatedmacular degeneration and also cataracts in an animal model (Marneros AG,EMBO Molecular Medicine, 2016; 8 (3): 208). Thus, in some embodiments,dendrimers are conjugated to one or more active agents effective inreducing the quantity and/or activity of one or more of VEGF-A, VEGF-B,VEGF-C, VEGF-D, and placental growth factor (PlGF).

Most notable angiogenesis inhibitors target the vascular endothelialgrowth factor signaling pathway, such as the monoclonal antibodybevacizumab (AVASTIN®, Genentech/Roche) and two kinase inhibitorssunitinib (SU11248, SUTENT®, Pfizer) and sorafenib (BAY43-9006,NEXAVAR®, Bayer). Bevacizumab was the first angiogenesis inhibitor thatwas clinically approved, initially for treatment of colorectal cancerand recently also for breast cancer and lung cancer. Another anti-VEGFagent clinically available is pegaptanib sodium, an aptamer forneovascular AMD. Unlike bevacizumab, which binds all VEGF isoforms,pegaptanib targets only VEGF165, the isoform responsible forpathological ocular neovascularization. In some embodiments, dendrimersare conjugated to one or more VEGF inhibitors including bevacizumab andpegaptanib sodium.

The small-molecule tyrosine kinase inhibitors sunitinib and sorafenibtarget the VEGF receptor (VEGFR), primarily VEGFR-2. Both drugs haveshown benefit in patients with renal cell cancer (Motzer R J, Bukowski RM, J Clin Oncol. (2006); 24(35):5601-8). Sunitinib is a potent inhibitorof angiogenesis, with a rabbit model of corneal neovascularizationsuggesting topical sunitinib is almost three times as effective asbevacizumab (Perez-Santonja J J et al., Am J Ophthalmol. 2010 October;150(4):519-528). Sorafenib inhibits Raf serine kinase. Cediranib is anoral tyrosine kinase inhibitor of VEGF receptor (VEGFR).

In some embodiments, dendrimers are conjugated to one or more VEGFreceptor inhibitors including Sunitinib (SU11248; SUTENT®), Sorafenib(BAY439006; NEXAVAR®), Pazopanib (GW786034; VOTRIENT®), Vandetanib(ZD6474; ZACTIMA®), Axitinib (AG013736), Cediranib (AZD2171; RECENTIN®),Vatalanib (PTK787; ZK222584), Dasatinib, Nintedanib, and Motesanib(AMG706). In preferred embodiments, the VEGF receptor inhibitors can befunctionalized with one or more spacers/linkers, for example with ether,ester, or amide linkage, for ease of conjugation with the dendrimersand/or for desired release kinetics. For example, sunitinib can bemodified to sunitinib with an ester linkage, or with an amide linkage(FIGS. 1A and 1B). Exemplary conjugation of a VEGF receptor inhibitor,e.g., sunitinib to a dendrimer is shown in FIG. 1A (via a hydroxymethyllinkage) and FIG. 1B (via an amide linkage). In preferred embodiments,the conjugation of dendrimer and/or one or more linkers does notsignificantly affect the activities of the active agents. In furtherpreferred embodiments, a VEGF receptor inhibitor is conjugated todendrimers with or without a spacer in such a way that it minimizes thereduction in inhibition, for example, less than 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, and100-fold. For example in the case of sunitinib, it retains its bindingaffinity towards one or more of VEGFR TKR after conjugation todendrimers at a level comparable to unconjugated sunitinib.

Additional VEGF receptor inhibitors with a functional spacer/linkage areshown below.

Structure II a-b: Chemical Structures of Sorafenib Analogues

Structure III a-d: Chemical Structure of Nintedanib Analogue 1

Structure IV: Chemical Structure of Orantinib Analogue

Orantinb-Amide-Linker Azide

In some embodiments, the dendrimer complexes including one or more VEGFreceptor inhibitors are administered in an amount effective to reduce orinhibit endothelial cells angiogenesis and/or vascular endothelial cellproliferation, to reduce retinal and/or choroidal angiogenesis and/or torelieve to one or more of the symptoms associated with the disease ordisorder of the eye.

TIE II Antagonists

In some embodiments, the dendrimers are complexed or conjugated with oneor more inhibitors of TIE II. Angiopoietin-1 receptor, also known asCD202B (cluster of differentiation 202B) and TIE II, is a protein thatin humans is encoded by the TEK gene. TIE2 is an angiopoietin receptor.The angiopoietins are protein growth factors required for the formationof blood vessels (angiogenesis), which supports tumor growth anddevelopment. Therefore, in some embodiments, dendrimers are conjugatedto one or more TIE II antagonists.

In some embodiments, the active agents are inhibitors of TIE II receptortyrosine kinase. Exemplary inhibitors of VEGFR/TIE II include CEP-11981and rebastinib. The TIE II antagonists can be functionalized, forexample with ether, ester, ethyl, or amide linkage, for ease ofconjugation with the dendrimers and/or for desired release kinetics. Thechemical structure of an exemplary TIE II antagonist is shown below asStructure XXI. TIE II inhibition of the free TIE II antagonist(Structure V) has a dissociation constant, K_(d), about 8.8 nm and theTIE II inhibition of dendrimer conjugated TIE II antagonist (StructureXXI) has a dissociation constant, K_(d), about 25 nm. Thus, in preferredembodiments, TIE II antagonists are conjugated to dendrimers with orwithout a spacer in such a way that it minimizes the reduction in TIE IIinhibition, for example, less than 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, and 100-fold. Inpreferred embodiments, the active agents are inhibitor of vascularendothelial growth factor receptor (VEGFR) and TIE II receptor tyrosinekinases.

Structure V: TIE II Antagonist 1

Anti-Inflammatory Agents

In some embodiments, one or more active agents associated with orcomplexed to dendrimers are one or more anti-inflammatory agents.Anti-inflammatory agents reduce inflammation and include steroidal andnon-steroidal drugs. Suitable steroidal active agents includeglucocorticoids, progestins, mineralocorticoids, and corticosteroids. Insome embodiments, one or more active agents are one or morecorticosteroids.

Exemplary anti-inflammatory agents include triamcinolone acetonide,fluocinolone acetonide, methylprednisolone, prednisolone, dexamethasone,loteprendol, fluorometholone, ibuprofen, aspirin, and naproxen.Exemplary immune-modulating drugs include cyclosporine, tacrolimus andrapamycin. Exemplary non-steroidal anti-inflammatory drugs (NSAIDs)include mefenamic acid, aspirin, Diflunisal, Salsalate, Ibuprofen,Naproxen, Fenoprofen, Ketoprofen, Deacketoprofen, Flurbiprofen,Oxaprozin, Loxoprofen, Indomethacin, Sulindac, Etodolac, Ketorolac,Diclofenac, Nabumetone, Piroxicam, Meloxicam, Tenoxicam, Droxicam,Lornoxicam, Isoxicam, Meclofenamic acid, Flufenamic acid, Tolfenamicacid, elecoxib, Rofecoxib, Valdecoxib, Parecoxib, Lumiracoxib,Etoricoxib, Firocoxib, Sulphonanilides, Nimesulide, Niflumic acid, andLicofelone. In preferred embodiments, the active agent is triamcinoloneacetonide, prednisolone, dexamethasone, or analogues thereof. Exemplaryanalogues of triamcinolone acetonide, prednisolone, and dexamethasoneare shown below.

Structure VI a-f: Chemical Structure of Analogues of TriamcinoloneAcetonide, Prednisolone, Dexamethasone

In some embodiments, the active agent is N-acetyl-L-cysteine, or aderivative or analogue or prodrug thereof. In a preferred embodiment,N-acetyl-L-cysteine is conjugated to a hydroxyl-terminated PAMAMdendrimer via non-cleavable linkage for minimal release of freeN-acetyl-cysteine after in vivo administration. The synthesis route foran exemplary non-releasable (or non-cleavable) form of thedendrimer/N-acetyl-cysteine complexes is shown in FIG. 14. In oneembodiment, the dendrimer complex is dendrimer-NAC-carboxymethylatedconjugate as shown in FIG. 14. The non-releasable form of thedendrimer/N-acetyl-cysteine complex provides enhanced therapeuticefficacy as compared to a releasable or cleavable form of thedendrimer/N-acetyl-cysteine complex, for example, N-acetyl-L-cysteineconjugated to a hydroxyl-terminated PAMAM dendrimer via an esterlinkage.

In some embodiments, one or more active agents are polysialic acid(e.g., low molecular weight polySia with an average degree ofpolymerization 20 (polySia avDP20)), Translocator Protein Ligands (e.g.,Diazepam binding inhibitor (DBI)), Interferon-β (IFN-β), andminocycline.

In some cases, one or more active agents are anti-infective agents.Exemplary anti-infectious agents include antiviral agents, antibacterialagents, antiparasitic agents, and anti-fungal agents. Exemplaryantibiotics include moxifloxacin, ciprofloxacin, erythromycin,levofloxacin, cefazolin, vancomycin, tigecycline, gentamycin,tobramycin, ceftazidime, ofloxacin, gatifloxacin; antifungals:amphotericin, voriconazole, natamycin.

Diagnostic Agents

Dendrimer nanoparticles can include diagnostic agents useful fordetermining the location of administered particles. These agents canalso be used prophylactically. In some embodiments, dendrimers areconjugated to one or more diagnostic agents including indocyanine green,fluorescein (e.g., fluorescein isocyanate), boron-dipyrromethene,rhodamine, and rose Bengal. In preferred embodiments, the diagnosticagent is indocyanine green as shown below:

Structure VII: Chemical Structure of Indocyanine Green

Additional examples of diagnostic agents include paramagnetic molecules,fluorescent compounds, magnetic molecules, and radionuclides, x-rayimaging agents, and contrast media. Examples of other suitable contrastagents include gases or gas emitting compounds, which are radioopaque.Dendrimer complexes can further include agents useful for determiningthe location of administered compositions. Agents useful for thispurpose include fluorescent tags, radionuclides and contrast agents.

Exemplary diagnostic agents include dyes such as fluorescent dyes andnear infra-red dyes, SPECT imaging agents, PET imaging agents andradioisotopes. Representative dyes include carbocyanine,indocarbocyanine, oxacarbocyanine, thüicarbocyanine and merocyanine,polymethine, coumarine, rhodamine, xanthene, fluorescein,boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680,VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700,AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780,DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, andADS832WS.

Exemplary SPECT or PET imaging agents include chelators such asdiethylene tri-amine penta-acetic acid (DTPA),1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA),di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine(MAG3), and hydrazidonicotinamide (HYNIC).

Exemplary isotopes include Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Gd3+,Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, F-18,Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, and Dy-166.

In preferred embodiments, the dendrimer complex includes one or moreradioisotopes suitable for positron emission tomography (PET) imaging.Exemplary positron-emitting radioisotopes include carbon-11 (¹¹C),copper-64 (⁶⁴Cu), nitrogen-13 (¹³N), oxygen-15 (¹⁵O), gallium-68 (⁶⁸Ga),and fluorine-18 (¹⁸F), e.g., 2-deoxy-2-¹⁸F-fluoro-β-D-glucose (¹⁸F-FDG).

In preferred embodiments, the one or more diagnostic agents can befunctionalized with one or more spacers/linkers, for example with ether,ester, or amide linkage, for ease of conjugation with the dendrimersand/or for desired release kinetics.

In further embodiments, a singular dendrimer complex composition cansimultaneously treat, and/or diagnose a disease or a condition at one ormore locations in the body.

III. Pharmaceutical Formulations

Pharmaceutical compositions including one or more dendrimer complexesmay be formulated in a conventional manner using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically. Proper formulation is dependent uponthe route of administration chosen. Pharmaceutical formulations containone or more dendrimer complexes in combination with one or morepharmaceutically acceptable excipients. Representative excipientsinclude solvents, diluents, pH modifying agents, preservatives,antioxidants, suspending agents, wetting agents, viscosity modifiers,tonicity agents, stabilizing agents, and combinations thereof. Suitablepharmaceutically acceptable excipients are preferably selected frommaterials which are generally recognized as safe (GRAS), and may beadministered to an individual without causing undesirable biologicalside effects or unwanted interactions.

In preferred embodiments, the compositions are formulated for parenteraldelivery to the eye. In some embodiments, the compositions areformulated for subcutaneous or intravitreal injection. Typically thecompositions will be formulated in sterile saline or buffered solutionfor injection into the tissues or cells to be treated. The compositionscan be stored lyophilized in single use vials for rehydrationimmediately before use. Other means for rehydration and administrationare known to those skilled in the art. Remington's PharmaceuticalSciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000,p. 704, provides suitable formulations and examples of ophthalmic drugsadministered in the form of a pharmaceutically acceptable salt includetimolol maleate, brimonidine tartrate, and sodium diclofenac.

The compositions are preferably formulated in dosage unit form for easeof administration and uniformity of dosage. The phrase “dosage unitform” refers to a physically discrete unit of conjugate appropriate forthe patient to be treated. It will be understood, however, that thetotal single administration of the compositions will be decided by theattending physician within the scope of sound medical judgment. Thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models, usually mice, rabbits, dogs, orpigs. The animal model is also used to achieve a desirable concentrationrange and route of administration. Such information should then beuseful to determine useful doses and routes for administration inhumans. Therapeutic efficacy and toxicity of conjugates can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose is therapeutically effectivein 50% of the population) and LD50 (the dose is lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiescan be used in formulating a range of dosages for human use.

Pharmaceutical compositions formulated for administration by parenteral(intramuscular, intraperitoneal, intravenous or subcutaneous injection)and enteral routes of administration are described. In preferredembodiments, the compositions are administered via a systemicadministration. In one embodiment, the compositions are administered viasubcutaneous route. In another embodiment, the compositions areadministered orally.

A. Parenteral Administration

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and includewithout limitation intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion. Insome embodiments, the dendrimers are administered parenterally, forexample, by subdural, intravenous, intrathecal, intraventricular,intraarterial, intra-amniotic, intraperitoneal, or subcutaneous routes.In preferred embodiments, the dendrimer compositions are administeredvia subcutaneous injection.

For liquid formulations, pharmaceutically acceptable carriers may be,for example, aqueous or non-aqueous solutions, suspensions, emulsions oroils. Parenteral vehicles include, for example, sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's and fixed oils. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, and injectable organic esters such as ethyloleate. Aqueous carriers include, for example, water, alcoholic/aqueoussolutions, cyclodextrins, emulsions or suspensions, including saline andbuffered media. The dendrimers can also be administered in an emulsion,for example, water in oil. Examples of oils are those of petroleum,animal, vegetable, or synthetic origin, for example, peanut oil, soybeanoil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil,cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fattyacids for use in parenteral formulations include, for example, oleicacid, stearic acid, and isostearic acid. Ethyl oleate and isopropylmyristate are examples of suitable fatty acid esters.

Formulations suitable for parenteral administration can includeantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Intravenous vehicles can include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose. Ingeneral, water, saline, aqueous dextrose and related sugar solutions,and glycols such as propylene glycols or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

Injectable pharmaceutical carriers are well-known to those of ordinaryskill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B.Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15thed., pages 622-630 (2009)).

B. Enteral Administration

In some embodiments, the compositions are formulated to be administeredenterally. The carriers or diluents may be solid carriers such ascapsule or tablets or diluents for solid formulations, liquid carriersor diluents for liquid formulations, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may be,for example, aqueous or non-aqueous solutions, suspensions, emulsions oroils. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, and injectable organic esters such as ethyl oleate.Aqueous carriers include, for example, water, alcoholic/aqueoussolutions, cyclodextrins, emulsions or suspensions, including saline andbuffered media.

Examples of oils are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, mineral oil, olive oil,sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil,olive, petrolatum, and mineral. Suitable fatty acids for use inparenteral formulations include, for example, oleic acid, stearic acid,and isostearic acid. Ethyl oleate and isopropyl myristate are examplesof suitable fatty acid esters.

Vehicles include, for example, sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Formulations include, for example, aqueous and non-aqueous,isotonic sterile injection solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. Vehicles can include,for example, fluid and nutrient replenishers, electrolyte replenisherssuch as those based on Ringer's dextrose. In general, water, saline,aqueous dextrose and related sugar solutions are preferred liquidcarriers. These can also be formulated with proteins, fats, saccharidesand other components of infant formulas.

In preferred embodiments, the compositions are formulated for oraladministration. Oral formulations may be in the form of chewing gum, gelstrips, tablets, capsules or lozenges. Encapsulating substances for thepreparation of enteric-coated oral formulations include celluloseacetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate and methacrylic acid ester copolymers. Solidoral formulations such as capsules or tablets are preferred. Elixirs andsyrups also are well known oral formulations.

IV. Methods of Making

A. Methods of Making Dendrimers

Dendrimers can be prepared via a variety of chemical reaction steps.Dendrimers are usually synthesized according to methods allowingcontrolling their structure at every stage of construction. Thedendritic structures are mostly synthesized by two main differentapproaches: divergent or convergent.

In some embodiments, dendrimers are prepared using divergent methods, inwhich the dendrimer is assembled from a multifunctional core, which isextended outward by a series of reactions, commonly a Michael reaction.The strategy involves the coupling of monomeric molecules that possessesreactive and protective groups with the multifunctional core moietywhich leads to stepwise addition of generations around the core followedby removal of protecting groups. For example, PAMAM-NH2 dendrimers arefirst synthesized by coupling N-(2-aminoethyl) acryl amide monomers toan ammonia core.

In other embodiments, dendrimers are prepared using convergent methods,in which dendrimers are built from small molecules that end up at thesurface of the sphere, and reactions proceed inward building inward andare eventually attached to a core.

Many other synthetic pathways exist for the preparation of dendrimers,such as the orthogonal approach, accelerated approaches the Double-stageconvergent method or the hypercore approach, the hypermonomer method orthe branched monomer approach, the Double exponential method; theOrthogonal coupling method or the two-step approach, the two monomersapproach, AB₂—CD₂ approach.

In some embodiments, the core of the dendrimer, one or more branchingunits, one or more linkers/spacers, and/or one or more surface groupscan be modified to allow conjugation to further functional groups(branching units, linkers/spacers, surface groups, etc.), monomers,and/or active agents via click chemistry, employing one or moreCopper-Assisted Azide-Alkyne Cycloaddition (CuAAC), Diels-Alderreaction, thiol-ene and thiol-yne reactions, and azide-alkyne reactions(Arseneault M et al., Molecules. 2015 May 20; 20(5):9263-94). In someembodiments, pre-made dendrons are clicked onto high-density hydroxylpolymers. ‘Click chemistry’ involves, for example, the coupling of twodifferent moieties (e.g., a core group and a branching unit; or abranching unit and a surface group) via a 1,3-dipolar cycloadditionreaction between an alkyne moiety (or equivalent thereof) on the surfaceof the first moiety and an azide moiety (e.g., present on a triazinecomposition) (or equivalent thereof) (or any active end group such as,for example, a primary amine end group, a hydroxyl end group, acarboxylic acid end group, a thiol end group, etc.) on the secondmoiety.

In some embodiments, dendrimer synthesis replies upon one or morereactions selected from thiol-ene click reactions, thiol-yne clickreactions, CuAAC, Diels-Alder click reactions, azide-alkyne clickreactions, Michael Addition, epoxy opening, esterification, silanechemistry, and a combination thereof.

Any existing dendritic platforms can be used to make dendrimers ofdesired functionalities, i.e., with a high-density of surface hydroxylgroups by conjugating high-hydroxyl containing moieties such as1-thio-glycerol or pentaerythritol. Exemplary dendritic platforms suchas polyamidoamine (PAMAM), poly (propylene imine) (PPI), poly-L-lysine,melamine, poly (etherhydroxylamine) (PEHAM), poly (esteramine) (PEA) andpolyglycerol can be synthesized and explored.

Still further, suitable dendrimers can be prepared by combining two ormore dendrons. Dendrons are wedge-shaped sections of dendrimers withreactive focal point functional groups. Many dendron scaffolds arecommercially available. They come in 1, 2, 3, 4, 5, and 6th generationswith, respectively, 2, 4, 8, 16, 32, and 64 reactive groups. In certainexamples, one type of active agents are linked to one type of dendronand a different type of active agents are linked to another type ofdendron. The two dendrons are then connected to form a dendrimer. Thetwo dendrons can be linked via click chemistry i.e., a 1,3-dipolarcycloaddition reaction between an azide moiety on one dendron and alkynemoiety on another to form a triazole linker.

Exemplary methods of making dendrimers are described in detail inInternational Patent Publication Nos. WO2009/046446, WO2015168347,WO2016025745, WO2016025741, WO2019094952, and U.S. Pat. No. 8,889,101.

B. Dendrimer Complexes

Dendrimer complexes can be formed of therapeutically active agents orcompounds conjugated or attached to a dendrimer, a dendritic polymer ora hyperbranched polymer. Techniques for conjugation of one or moreactive agents to a dendrimer are known in the art, and are described indetail in U.S. Published Application Nos. US 2011/0034422, US2012/0003155, and US 2013/0136697.

In some embodiments, one or more active agents are covalently attachedto the dendrimers. In some embodiments, the active agents are attachedto the dendrimer via a linking moiety that is designed to be cleaved invivo. The linking moiety can be designed to be cleaved hydrolytically,enzymatically, or combinations thereof, so as to provide for thesustained release of the active agents in vivo. Both the composition ofthe linking moiety and its point of attachment to the active agent, areselected so that cleavage of the linking moiety releases either anactive agent, or a suitable prodrug thereof. The composition of thelinking moiety can also be selected in view of the desired release rateof the active agents.

In some embodiments, the attachment occurs via one or more of disulfide,ester, ether, thioester, carbamate, carbonate, hydrazine, or amidelinkages. In preferred embodiments, the attachment occurs via anappropriate spacer that provides an ester bond or an amide bond betweenthe agent and the dendrimer depending on the desired release kinetics ofthe active agent. In some cases, an ester bond is introduced forcleavable form of active agents. In other cases, an amide bond isintroduced for non-cleavable form of active agents. Exemplary synthesisroutes are described in Example 4 and 5 to show introduction ofnon-cleavable linkages between active agents and dendrimers.

Linking moieties generally include one or more organic functionalgroups. Examples of suitable organic functional groups include secondaryamides (—CONH—), tertiary amides (—CONR—), sulfonamide (—S(O)₂—NR—),secondary carbamates (—OCONH—; —NHCOO—), tertiary carbamates (—OCONR—;—NRCOO—), carbonate (—O—C(O)—O—), ureas (—NHCONH—; —NRCONH—; —NHCONR—,—NRCONR—), carbinols (—CHOH—, —CROH—), disulfide groups, hydrazones,hydrazides, ethers (—O—), and esters (—COO—, —CH₂O₂C—, CHRO₂C—), whereinR is an alkyl group, an aryl group, or a heterocyclic group. In general,the identity of the one or more organic functional groups within thelinking moiety can be chosen in view of the desired release rate of theactive agents. In addition, the one or more organic functional groupscan be chosen to facilitate the covalent attachment of the active agentsto the dendrimers. In preferred embodiments, the attachment can occurvia an appropriate spacer that provides a disulfide bridge between theagent and the dendrimer. The dendrimer complexes are capable of rapidrelease of the agent in vivo by thiol exchange reactions, under thereduced conditions found in body.

In certain embodiments, the linking moiety includes one or more of theorganic functional groups described above in combination with a spacergroup. The spacer group can be composed of any assembly of atoms,including oligomeric and polymeric chains; however, the total number ofatoms in the spacer group is preferably between 3 and 200 atoms, morepreferably between 3 and 150 atoms, more preferably between 3 and 100atoms, most preferably between 3 and 50 atoms. Examples of suitablespacer groups include alkyl groups, heteroalkyl groups, alkylarylgroups, oligo- and polyethylene glycol chains, and oligo- and poly(aminoacid) chains. Variation of the spacer group provides additional controlover the release of the anti-inflammatory agents in vivo. In embodimentswhere the linking moiety includes a spacer group, one or more organicfunctional groups will generally be used to connect the spacer group toboth the anti-inflammatory agent and the dendrimers.

Reactions and strategies useful for the covalent attachment of activeagents to dendrimers are known in the art. See, for example, March,“Advanced Organic Chemistry,” 5th Edition, 2001, Wiley-IntersciencePublication, New York) and Hermanson, “Bioconjugate Techniques,” 1996,Elsevier Academic Press, U.S.A. Appropriate methods for the covalentattachment of a given active agent can be selected in view of thelinking moiety desired, as well as the structure of the active agentsand dendrimers as a whole as it relates to compatibility of functionalgroups, protecting group strategies, and the presence of labile bonds.

The optimal drug loading will necessarily depend on many factors,including the choice of drug, dendrimer structure and size, and tissuesto be treated. In some embodiments, the one or more active drugs areencapsulated, associated, and/or conjugated to the dendrimer at aconcentration of about 0.01% to about 45%, preferably about 0.1% toabout 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 1% toabout 10%, about 1% to about 5%, about 3% to about 20% by weight, andabout 3% to about 10% by weight. However, optimal drug loading for anygiven drug, dendrimer, and site of target can be identified by routinemethods, such as those described.

In some embodiments, conjugation of active agents and/or linkers occursthrough one or more surface and/or interior groups. Thus, in someembodiments, the conjugation of active agents/linkers occurs via about1%, 2%, 3%, 4%, or 5% of the total available surface functional groups,preferably hydroxyl groups, of the dendrimers prior to the conjugation.In other embodiments, the conjugation of active agents/linkers occurs onless than 5%, less than 10%, less than 15%, less than 20%, less than25%, less than 30%, less than 35%, less than 40%, less than 45%, lessthan 50%, less than 55%, less than 60%, less than 65%, less than 70%,less than 75% total available surface functional groups of thedendrimers prior to the conjugation. In preferred embodiments, dendrimercomplexes retain an effective amount of surface functional groups fortargeting to specific cell types, whilst conjugated to an effectiveamount of active agents for treat, prevent, and/or image the disease ordisorder.

1. Dendrimer Conjugation to Active Agents Via Ether Linkages

A method to incorporate one or more active agents onto ahydroxyl-terminated dendrimer via an ether linkage, optionally via oneor more linkers/spacers, has been developed.

In some embodiments, surface or terminal groups of hydroxyl-terminateddendrimers are modified via etherification reaction prior to conjugationto one or more linkers/spacers and one or more radionuclides.Etherification is the dehydration of an alcohol to form ethers. In someembodiments, one or more hydroxyl groups of hydroxyl-terminateddendrimers undergo etherification reaction prior to conjugation to oneor more linking moieties and one or more active agents.

In some embodiments, ether linkage is introduced at the surface groupsof hydroxyl PAMAM dendrimer by reacting with propargyl bromide in thepresence of 2% sodium hydroxide solution in DMSO. In a furtherembodiment, etherification reaction of generation 4 hydroxyl-terminatedPAMAM dendrimer, PAMAM-G4-OH, using allyl bromide, anhydrous cesiumcarbonate and tetrabutylammonium iodide in DMF.

In other embodiments, at dendrimer generation 3.5, alkyne functionalgroups are introduced using a polyethyl glycol (PEG) linker with anamine at one end and a hexyne at the other end to produce a generation 4bifunctional dendrimer, i.e., with hydroxyl groups and ether linkagesready for further conjugation. An exemplary bifunctional dendrimer isshown as compound 1 in FIG. 11 with 7 alkyne arms and 57 hydroxyl groupson the surface.

V. Methods of Use

The dendrimer complex compositions are generally suitable for treatmentof one or more diseases or disorders associated with the eye,particularly inflammatory and/or angiogenic diseases in the eye.Dendrimer compositions and methods thereof for targeted delivery of oneor more active agents to the diseased tissues/cells in the eye viasystemic administration with increased efficacy and reduced side effectsare described, preferably via selectively targeting to affectedcells/tissue including activated microglia and activated macrophage,retinal pigment epithelia (RPE) cells, and/or choroidal neovascular(CNV) lesions. Preferably dendrimer compositions and methods thereof fortargeted delivery give rise to minimal dendrimer in non-injured regionof optic nerve or CNS. Methods for treating back of the eye disordersare also described. In some embodiments, the dendrimer complexes areused to treat exudative form of age-related macular degeneration (AMD).The methods typically include administering a subject in a need thereofan effective amount of a composition including dendrimer and one or moreactive agents.

Methods of reducing and/or inhibiting the number or activities ofactivated microglia and macrophages in the retina and/or the choroid inthe eye of a subject in need thereof are provided. In some embodiments,treatment using an effective amount of the compositions includinghydroxyl-terminated dendrimer complexed, covalently conjugated orintra-molecularly dispersed or encapsulated with one or more therapeuticagents is administered to reduce and/or inhibit the number or activitiesof the activated microglia and macrophages in the retina and/or thechoroid in the eye in need thereof. In some embodiments, thecompositions are administered in a dosage and via a route to inhibit orreduce activation of microglia in the retina. In other embodiments,compositions can inhibit or reduce phagocytic activities of microglia.In other embodiments, compositions including one or more receptortyrosine kinase inhibitors can inhibit or reduce activity and/orquantity of activated microglia and macrophages in the diseased retinaand/or choroid of a subject by about 10%, 20%, 30%, 40%, 50%, 75%, 85%,90%, 95%, or 99% compared to the activity and/or quantity of the samecells in equivalent diseased tissues of subjects that did not receive,or were not treated with the dendrimer compositions (e.g., un-conjugatedactive agents).

Methods of reducing and/or inhibiting the expression and/or activitiesof VEGF and/or VEGFR in the activated microglia, activated macrophages,and/or retinal pigment epithelial (RPE) cells in the diseased retinaand/or choroid are also described. In some embodiments, the compositionsare applied via systemic routes such as intravenous injections,subcutaneous injections or oral administration. In preferredembodiments, the compositions are not administered intravitreally orsubchoroidally, which can result in direct damage and/or inflammation tothe eye. Methods of reducing and/or inhibiting one or morepro-inflammatory cytokines secreted by the activated microglia andmacrophages in the diseased retina and/or choroid are also described. Insome embodiments, treatment using an effective amount of thecompositions leads to a decrease in expression of one or morepro-inflammatory cytokines (e.g., TNF-α, interleukin-1β (IL-1β), orinterferon-γ (IFN-γ)) secreted by the activated microglia andmacrophages in the diseased retina and/or choroid by about 10%, 20%,30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% compared to those inequivalent diseased tissues of subjects that did not receive, or werenot treated with the dendrimer compositions (e.g., un-conjugated activeagents).

Methods of reducing and/or inhibiting one or more pro-oxidativeproperties of the activated microglia and macrophages in the diseasedretina and/or choroid are also described. In some embodiments, treatmentusing an effective amount of the compositions leads to a decrease in theoxidative stress of the activated microglia and macrophages in thediseased retina and/or choroid, for example by reducing nitric oxide(NO) production or inducible nitric oxide synthase (iNOS) activation(e.g., NOS2 expression), by about 10%, 20%, 30%, 40%, 50%, 75%, 85%,90%, 95%, or 99% compared to those in equivalent diseased tissues ofsubjects that did not receive, or were not treated with the dendrimercompositions (e.g., un-conjugated active agents).

Methods of reducing and/or inhibiting abnormal vascular permeability andleakage, and/or neoangiogenesis in the eye of a subject in need thereofare also described. In some embodiments, treatment using an effectiveamount of the compositions leads to a decrease in vascular leakageand/or neoangiogenesis.

A. Treatment Regimen

1. Dosage and Effective Amounts

Dosage and dosing regimens are dependent on the severity and location ofthe disorder or injury and/or methods of administration, and are knownto those skilled in the art. A therapeutically effective amount of thedendrimer composition used in the treatment of one or more eye diseasesis typically sufficient to treat, inhibit, or alleviate one or moresymptoms associated with the eye.

In some in vivo approaches, the dendrimer complexes are administered toa subject in a therapeutically effective amount to reduce or inhibitocular angiogenesis, particularly retinal and choroidalneovascularization. In some embodiments, an effective amount of thecomposition is used to reduce or inhibit endothelial cells angiogenesisand/or vascular endothelial cell proliferation.

A pharmaceutical composition including a therapeutically effectiveamount of the dendrimer compositions and a pharmaceutically acceptablediluent, carrier or excipient is described. In some embodiments, thepharmaceutical composition includes an effective amount ofhydroxyl-terminated dendrimers conjugated to a VEGF receptor tyrosinekinase inhibitor. In some particular embodiments, dosage ranges suitablefor parenteral use are between about 0.1 mg/kg and about 200 mg/kg,inclusive; between about 0.5 mg/kg and about 100 mg/kg, inclusive;between about 1.0 mg/kg and about 40 mg/kg, inclusive; and between about2.0 mg/kg and about 20 mg/kg, inclusive. Higher doses may be giveninitially to load the patient with drug and maximize uptake in thediseased tissues (e.g. eye). After the loading dose, patients mayreceive a maintenance dose. Loading doses may range from 10 to 100 mg/kgof body weight and maintenance doses may range from 0.1 to <10 mg/kg ofbody weight. When administered enterally, the dose required fortreatment may be up to 10 fold greater than the effective parenteraldose. The optimal dose is selected from the safety and efficacy resultsof each tested dose for each drug in patients.

Dosage forms of the pharmaceutical composition including the dendrimercompositions are also provided. “Dosage form” refers to the physicalform of a dose of a therapeutic compound, such as a capsule or vial,intended to be administered to a patient. The term “dosage unit” as usedherein refers to the amount of the therapeutic compounds to beadministered to a patient in a single dose. In some embodiments, thedosage unit suitable for use are (assuming the weight of an averagepatient being 70 kg) between 5 mg/dosage unit and about 14,000 mg/dosageunit, inclusive; between about 35 mg/dosage unit and about 7,000mg/dosage unit, inclusive; and between about 70 mg/dosage unit and about2,800 mg/dosage unit, inclusive; and between about 140 mg/dosage unitand about 1,400 mg/dosage unit, inclusive.

The actual effective amounts of dendrimer complex can vary according tofactors including the specific active agent administered, the particularcomposition formulated, the mode of administration, and the age, weight,condition of the subject being treated, as well as the route ofadministration and the disease or disorder.

Preferably the dendrimer compositions including one or more activeagents, for example sunitinib, are delivered to cells in and around thediseases or injured tissues, (e.g. microglia). For example, dendrimercomplex compositions can be in an amount effective to deliver one ormore active agents to cells at or nearby the site of inflammation,particularly inflammation of the eye. Therefore, in some embodiments,the dendrimer complex compositions including one or more active agentare in an amount effective to ameliorate inflammation in a subject. In apreferred embodiment, the effective amount of dendrimer complexcompositions does not induce significant cytotoxicity in the cells of asubject compared to an untreated control subject. Preferably, the amountof dendrimer complex compositions is effective to prevent or reduceinflammation and/or further associated symptoms of a disease or disorderin a subject compared to an untreated control.

In general, the timing and frequency of administration will be adjustedto balance the efficacy of a given treatment or diagnostic schedule withthe side-effects of the given delivery system. Exemplary dosingfrequencies include continuous infusion, single and multipleadministrations such as hourly, daily, weekly, monthly or yearly dosing.

In some embodiments, dosages are administered once, twice, or threetimes daily, or less frequently, for example, every other day, two days,three days, four days, five days, or six days to a human. In someembodiments, dosages are administered only about once or twice everyweek, every two weeks, every three weeks, or every four weeks. In someembodiments, dosages are administered about once or twice every month,every two months, every three months, every four months, every fivemonths, or every six months, or less frequent. In a preferredembodiment, dosages are administered once every four weeks or lessfrequent.

It will be understood by those of ordinary skill that a dosing regimencan be any length of time sufficient to treat the disorder in thesubject. In some embodiments, the regimen includes one or more cycles ofa round of therapy followed by a drug holiday (e.g., no drug). The roundof the therapy can be, for example, and of the administrations discussedabove. Likewise, the drug holiday can be 1, 2, 3, 4, 5, 6, or 7 days; or1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

2. Controls

The therapeutic result of the dendrimer complex compositions includingone or more active agents can be compared to a control. Suitablecontrols are known in the art and include, for example, untreated cellsor an untreated subject. A typical control is a comparison of acondition or symptom of a subject prior to and after administration ofthe targeted agent. The condition or symptom can be a biochemical,molecular, physiological, or pathological readout. For example, theeffect of the composition on a particular symptom, pharmacologic, orphysiologic indicator can be compared to an untreated subject, or thecondition of the subject prior to treatment. In some embodiments, thesymptom, pharmacologic, or physiologic indicator is measured in asubject prior to treatment, and again one or more times after treatmentis initiated. In some embodiments, the control is a reference level, oraverage determined based on measuring the symptom, pharmacologic, orphysiologic indicator in one or more subjects that do not have thedisease or condition to be treated (e.g., healthy subjects). In someembodiments, the effect of the treatment is compared to a conventionaltreatment that is known the art.

B. Subjects to be Treated

The compositions and methods are suitable for treatment one or morediseases or disorders of the eye. The compositions and methods aresuitable for alleviating one or more symptoms associated with one ormore diseases or disorder of the eye, for example, discomfort, pain,dryness, excessive tearing, injuries, infections, burns, and gradualloss of vision.

In some embodiments, the eye disorder to be treated is a back of the eyedisease such as diabetic eye disease, symptomatic vitreomacularadhesion/vitreomacular traction (sVMA/VMT), and wet (neovascular) or dryAMD (age-related macular degeneration). In some embodiments, the eyedisorder to be treated is one or more retinal and choroidal vasculardiseases (e.g., AMD, retinopathy of prematurity, diabetic macular edema,retinal vein occlusion, retinopathy associated with toxicity ofchemotherapy e.g., MEK retinopathy). In preferred embodiments, the eyedisorder to be treated is age-related macular degeneration (AMD).Age-related macular degeneration (AMD) is a neurodegenerative,neuroinflammatory disease of the macula, which is responsible forcentral vision loss. The pathogenesis of age-related maculardegeneration involves chronic neuroinflammation in the choroid (a bloodvessel layer under the retina), the retinal pigment epithelium (RPE), acell layer under the neurosensory retina, Bruch's membrane and theneurosensory retina, itself.

In other embodiments, the eye disorder to be treated is an inflammatorydisease of the eye, i.e., diseases of the eye associated withinflammation of the tissues of the eye, including, for example, AMD,retinitis pigmentosa, optic neuritis, sarcoid, retinal detachment,temporal arteritis, retinal ischemia, arteriosclerotic retinopathy,hypertensive retinopathy, retinal artery blockage, retinal veinblockage, diabetic retinopathy, macular edema, Stargardt disease (alsoknown as Stargardt macular dystrophy or juvenile macular degeneration),geographic atrophy, neuromyelitis optica, and also including angiogenicdiseases including, for example, retinal neovascularization andchoroidal neovascularization. Other conditions can also result ininflammation and/or angiogenesis in the eye, for example, infection,sickle cell disease, hypotension, etc.

Further examples of eye disorders that may be treated include amoebickeratitis, fungal keratitis, bacterial keratitis, viral keratitis,onchorcercal keratitis, bacterial keratoconjunctivitis, viralkeratoconjunctivitis, corneal dystrophic diseases, Fuchs' endothelialdystrophy, meibomian gland dysfunction, anterior and posteriorblepharitis, conjunctival hyperemia, conjunctival necrosis, cicatricalscaring and fibrosis, punctate epithelial keratopathy, filamentarykeratitis, corneal erosions, thinning, ulcerations and perforations,Sjogren's syndrome, Stevens-Johnson syndrome, autoimmune dry eyediseases, environmental dry eye diseases, corneal neovascularizationdiseases, post-corneal transplant rejection prophylaxis and treatment,autoimmune uveitis, infectious uveitis, anterior uveitis, posterioruveitis (including toxoplasmosis), pan-uveitis, inflammatory disease ofthe vitreous or retina, endophthalmitis prophylaxis and treatment,macular edema, macular degeneration, age-related macular degeneration,proliferative and non-proliferative diabetic retinopathy, hypertensiveretinopathy, an autoimmune disease of the retina, primary and metastaticintraocular melanoma, other intraocular metastatic tumors, open angleglaucoma, closed angle glaucoma, pigmentary glaucoma and combinationsthereof. Other disorders include injury, burn, or abrasion of thecornea, cataracts and age related degeneration of the eye or visionassociated therewith.

The dendrimer complexes can be administered in combination with one ormore additional therapeutically active agents, which are known to becapable of treating conditions or diseases discussed above.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Examples Example 1: Targeted Sustained Intracellular Delivery toChoroidal Neovascular Lesions after a Single Systemic Administration asDemonstrated by Imaging

Methods

Hydroxyl dendrimers (˜14000 Da) were covalently conjugated to 2-3indocyanine green (ICG) molecules (D-ICG) per dendrimer vianon-cleavable linkages. Hydroxyl dendrimers (˜14000 Da) were covalentlyconjugated to 2-3 tetramethylrhodamine (TRITC) molecules (D-TRITC) perdendrimer via non-cleavable linkages.

Two studies were conducted in C57BL/6 mice (n=5/group) administeredintravenously 100 μL of D-ICG or vehicle control. In the first study,mice were administered D-ICG or vehicle control at 1, 3, 7, or 14 dayspost-laser and eyes were analyzed by optical coherence tomography (OCT)with ICG imaging at 4 or 24 hr post-dose. Flat-mounts of thesclera-choroid/retinal pigment epithelial (RPE) complexes were stainedby fluorescently tagged isolectin and IBA-1.

The second study evaluated the localization and persistence of dendrimerconjugates in the CNV lesion. In the second study, mice wereadministered intravenously 100 μL of D-ICG and 100 μL of D-TRITC (1 hrafter D-ICG) or vehicle control at 24 hr post-laser. Mice analyzed andsacrificed at 4, 7, 14, 21, and 28 days post-dose (n=5/group). Forcontrol group, free ICG (1.23 mg/mL), 100 μL, IV dosed 24 hr post-laserand mice analyzed and sacrificed at 2, 4, 7, and 14 days post-dose(n=5/group). Eyes analyzed by optical coherence tomography (OCT) withICG imaging. Flat-mounts of the sclera-choroid/retinal pigmentepithelial (RPE) complexes were stained by fluorescently tagged IBA-1alone.

Results

No significant release of ICG or TRITC was observed from dendrimersunder in vitro release when was evaluated at 37° C. in PBS, pH 7.4 orcitrate buffer, pH 5.5 with esterase.

The ability of hydroxyl dendrimers labelled with indocyanine green (ICG)was evaluated to target choroidal neovascular (CNV) lesions, and furtherinto macrophages and the retinal pigment epithelium, after systemicadministration in a mouse model of laser-induced CNV.

Systemically administered D-ICG was selectively taken up by cells withinthe CNV lesions within 24 hr after dosing, whereas the free ICGdistributes non-specifically and is typically cleared within hours.Reactive macrophage and microglia endocytosed dendrimer conjugates 24 hrpost-laser and lesion showed greater uptake during early stage of CNV,consistent with efficacy studies (24 hr post-laser). Dendrimerconjugates localized in macrophages in CNV lesions as shown byco-localization with IBA-1 positive cells (data not shown). Free ICGcontrol groups showed that free ICG no longer present in the lesionsbetween 7-14 days post-laser. IBA-1 signal increased up to 24-48 hrafter laser injury and isolectin increases slightly later at the 48 hpost-laser (FIGS. 1A and 2B). A single systemic D-ICG dose given 24 hrafter laser injury localized to the CNV lesion and significant D-ICG wasstill present at the last time point, i.e., 28 days (FIG. 2C).

Hydroxyl dendrimers co-localized with reactive macrophages in choroids,microglia/macrophages in retina, and RPE cells at the site ofinflammation/neovascularization. D-ICG and D-TRITC appeared to beintracellular and focused in regions of IBA-1 signal consistent withprevious studies demonstrating hydroxyl dendrimer uptake in reactivemicroglia, macrophage, and RPE cells.

Hydroxyl dendrimers (D-ICG) selectively target to CNV lesions aftersystemic administration and persist for at least 28 days post-dose,despite the hydroxyl dendrimers are systemically cleared within 48 hr.Thus, the hydroxyl dendrimer provided a prolonged localization at theCNV lesions suitable for sustained and targeted therapies, for example,once per month systemic (subcutaneous or oral) treatment for retinaldiseases with minimal systemic exposure.

Example 2: Suppression of Murine Choroidal Neovascularization afterSystemic Administration of a Targeted Anti-VEGF Therapy

Methods

Hydroxyl dendrimers (˜14000 Da), which selectively target inflammation,were covalently conjugated to analogs of sunitinib, an FDA-approvedpotent VEGF receptor tyrosine kinase inhibitor. Conjugates were madewith a cleavable sunitinib analog (D-CSA, compound 6 in FIG. 1A) or anon-cleavable sunitinib analog (D-NSA, compound 3 in FIG. 1B) and drugrelease was evaluated at 37° C. in PBS, pH 7.4 or citrate buffer, pH 5.5with esterase. Laser-induced rupture of Bruch's membrane was performedin both eyes of C57BL/6 mice (n=8/group) 24 hr prior to doseadministration. Mice were administered intravenously (IV, 100 μL)vehicle, D-CSA (5.25 (low) or 26.25 (high) mg/kg sunitinib equivalent),D-NSA (6.3 (low) or 15.75 (high) mg/kg sunitinib equivalent), or freesunitinib (32.5 mg/kg). As a positive control group, a cohort of micewere administered aflibercept (EYLEA®) intravitreally (IVT; 1 μL, 40μg). The CNV area was measured 7 days after laser treatment by bothfluorescein angiography and flat-mounts of the sclera-choroid/RPEcomplexes stained with isolectin IB4.

Results

The efficacy of hydroxyl dendrimers covalently conjugated with analogsof sunitinib was evaluated in a mouse model of laser-induced choroidalneovascularization (CNV).

D-CSA was prepared with 5 sunitinib analogs per dendrimer (10.5% w/w)and D-NSA was prepared with 7 sunitinib analogs per dendrimer (12.6%w/w). Over 6 days in vitro, D-CSA released ˜65% of the sunitinib at pH5.5 with esterase (intracellular conditions) and ˜15% release ofsunitinib occurred over 24 hr at pH 7.4 (plasma conditions). Release ofthe sunitinib analog from D-NSA conjugate is minimal.

Statistically significant reductions in the CNV area were observed forIVT aflibercept and both IV dose levels of D-CSA and D-NSA but not freesunitinib (even at 5-fold higher doses compared to low dose D-CSA),compared with vehicle control (FIG. 3).

Binding affinity (K_(d)) to VEGFR2 was assessed with free sunitinibmalate (0.13 nM), sunitinib analog attached via a non-cleavable PEGlinker (1 nM) and D-NSA (27 nM). The binding affinity data indicatedthat high binding affinity was retained in D-NSA. Thus, it has beendemonstrated that conjugation of a sunitinib analog to hydroxyldendrimers maintains nanomolar potency for VEGF RTK.

Single doses of D-CSA/D-NSA administered in laser-induced CNV mousemodel demonstrated efficacy comparable to aflibercept administeredintravitreally. The non-cleavable sunitinib analog efficacy in CNV areareduction suggests that sunitinib release from the dendrimer may not berequired. Previous studies have shown hydroxyl dendrimers anddendrimer-drug conjugates are retained in CNV lesions >28 days andsystemically cleared intact within 24 hr in mice and humans withoutdetectable liver or other off-target toxicity.

Example 3: Duration of Efficacy and Drug Systemic Clearance

Methods

Conjugates were made with a cleavable sunitinib analog (D-CSA, compound6 in FIG. 1A) or a non-cleavable sunitinib analog (D-NSA, compound 3 inFIG. 1B). Laser-induced rupture of Bruch's membrane was performed inboth eyes of C57BL/6 mice (n=8/group) 24 hr prior to doseadministration. Mice were administered a single intraperitonealinjection (IP, 100 μL) vehicle, D-CSA (5.25 mg/kg sunitinib equivalent),D-NSA (6.3 mg/kg sunitinib equivalent), or free sunitinib (6.5 mg/kg).As a positive control group, a cohort of mice were administeredaflibercept (EYLEA®) intravitreally (IVT; 1 μL, 40 μg). The CNV area wasmeasured 7 days and 14 days after laser treatment by both fluoresceinangiography and flat-mounts of the sclera-choroid/RPE complexes stainedwith isolectin IB4. For plasma pharmacokinetics study, the samedendrimer labeled with Cy5 administered via a single IP injection wasmonitored by plasma collection up to 72 hours after IP administration.

Results

The therapeutic duration and clearance of hydroxyl dendrimers covalentlyconjugated with analogs of sunitinib was evaluated in a mouse model oflaser-induced choroidal neovascularization (CNV).

Dendrimer conjugated sunitinib analog, D-CSA and D-NSA, demonstrated adurable response from a single IP dose with a reduction in the CNV areaat day 7 post-treatment and a further reductions in the CNV area at day14 post-treatment. Significant reduction in the CNV area was observedfor IVT aflibercept at day 7 post-treatment but the reduction was notsustained at day 14 post-treatment (FIG. 4A).

In the serum, both D-CSA and D-NSA were cleared within 2 dayspost-treatment (FIG. 4B). Thus, the dendrimer conjugated sunitinibanalog D-CSA and D-NSA provided a prolonged local effect on CNV lesionswith the lesion size continued to decrease at day 14 post-treatment.

Example 4: Synthesis and Characterization of N, N-Didesethyl SunitinibAmide Azide

The design and synthesis of dendrimer-didesethyl sunitinib conjugate isdescribed in Examples 4 and 5. Overexpression of vascular endothelialgrowth factor (VEGF) has been implicated in a number of diseasesassociated with angiogenesis. Sunitinib is a receptor tyrosine kinaseinhibitor that blocks VEGF receptors and has excellent antiangiogenicactivity and is approved by the FDA for use in different types ofcancers. Didesethyl sunitinib is an active metabolite of sunitinib.Despite the excellent therapeutic value of sunitinib and its analogues,their clinical development is hampered by the associated toxicity. Thedendrimer-didesethyl sunitinib conjugates aim to overcome the doserelated toxicities of sunitinib by attaching it to a hydroxyl terminateddendrimer. The synthesis scheme is outline in FIG. 5 and a generation 4PAMAM is used as an exemplary hydroxyl terminated dendrimer.

Step 1: Synthesis of 5-fluoro-2,3-dihydro-1H-indol-2-one (Compound 2)

To a stirred solution of 5-fluoro-2,3-dihydro-1H-indole-2,3-dione (6.0gm, 1.0 eq.) in n-butanol (10V) was added triethyl amine (6.12 mL, 1.2eq.) and followed by hydrazine hydrate (3.56 mL, 2.0 eq.) was added atroom temperature. The resulting solution was stirred for 16 hrs at 100°C. Reaction progress was monitored by TLC (50% ethylacetate in Hexanes).Once the reaction was judged to completion, reaction mass was as suchevaporated to dryness under vacuum at 45° C. to obtain dark brown solid.The obtained solid was quenched with water (20 V) and extracted withethyl acetate (30V) and organic layer was given water wash. Organiclayer was concentrated to dryness on rotary evaporator. The crudeproduct was purified by recrystallization using ethyl acetate to getgrey color fluffy solid. (4.0 g, 72% yield.) The compound 2 shown inFIG. 5 was confirmed by ¹H NMR, liquid chromatography, and massspectrometry.

Step 2: Synthesis of5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (Compound 4)

To a stirred solution of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound2) (4.0 gm, 1.0 eq.) and 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (compound 3) (4.41 gm, 1.0 eq.) in ethanol (10V) was addedpyrrolidine (4.42 mL, 2.0 eq.) at room temperature. The resultingsolution was stirred for 3 hrs at 80° C. Reaction progress was monitoredby TLC (10% Methanol in DCM). Once the reaction was judged tocompletion, reaction mass was cooled to room temperature added 2M HClsolution to pH=3. A brownish-red precipitate was formed and filtered.The obtained solid was washed with ethanol (20 V) followed by hexanes(30 V) and filtered to get reddish-orange solid. (6.6 g, 82% yield.) Thecompound 4 shown in FIG. 5 was confirmed by ¹H NMR.

Step 3: Synthesis of tert-butylN-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate(Compound 6)

To a solution of5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (compound 4) (6.5 g, 1.0 eq.) in DMF were added triethyl amine(6.08 mL, 2.0 eq.) EDC.HCl ((8.68 g, 2.1 eq.), HOBT (3.94 g, 1.35 eq.)and tert-butyl N-(2-aminoethyl) carbamate (4.16 g, 1.2 eq.) and at 0° C.Reaction was stirred at room temperature for 16 hrs. Reaction mixturewas diluted with water (20.0 V), stirred for 10 min. to precipitate andfiltered to get brown solid. The obtained solid was washed with ethylacetate (15.0 V), followed by hexanes (15.0V), filtered and dried to getbrownish-orange solid as a tert-butylN-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate(compound 6) (7.5 g, 78% yield). The compound 6 shown in FIG. 5 wasconfirmed by ¹H NMR.

Step 4: Synthesis ofN-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide(Compound 7)

To a solution of tert-butylN-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate(compound 6) (9.0 g, 1.0 eq.) in DCM (10.0 V) was added trifluoro aceticacid (3.0 V) at 0-5° C. Reaction was stirred at room temperature for 12hrs. Reaction mass was as such evaporated to dryness under vacuum at 45°C. to obtain dark brown solid. The obtained solid was washed withdiethyl ether (15.0 V) filtered and dried to get orange-yellow solid(6.0 g crude). The compound 7 shown in FIG. 5 was confirmed by ¹H NMR,liquid chromatography, and mass spectrometry.

Step 5: Synthesis ofN-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}-3-[2-(2-propoxyethoxy)ethoxy]propanamide(Compound 9)

To a solution of 3-[2-(2-propoxyethoxy)ethoxy]propanoic acid (8) (5.95g, 1.0 eq.) in DMF (10.0 V) were added DIPEA (8.40 mL, 2.0 eq.) EDC.HCl(6.90 g, 1.5 eq.), HOBT (0.65 g, 0.2 eq.),N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide(compound 7) (11.0 g, 1.0 eq.) and DMAP (0.294 g, 0.1 eq.) at 0-5° C.Reaction was stirred at room temperature for 3 hrs. Reaction progresswas monitored by TLC (10% MeOH in DCM). Reaction mixture was dilutedwith water (20.0 V) stirred for 10 min. to form brown precipitate andfiltered. The obtained solid purified by reverse phase columnchromatography to obtain N, N-didesethyl sunitinib amide azide as anorange solid (5.2 g, 37% yield). The compound 9 was confirmed by ¹H NMR,liquid chromatography, and mass spectrometry.

Example 5: Synthesis and Characterization of Dendrimer-DidesethylSunitinib Conjugate (D-4517)

Methods

Synthesis and Characterization of Intermediates and Dendrimer ConjugateSynthesis of Dendrimer Hexyne (Compound 2 of FIG. 6A)

Took a dried round bottom flask (250 mL) and recorded its empty weight.Poured the desired amount of the methanolic solution of G4-OH in theround bottom and evaporated at 60° C. for 2 hours. Shifted the flask onthe high vacuum assembly for 1 hour. Recorded the amount of G4-OH in theflask. Once the weight of G4-OH was recorded, added 50-60 mL ofanhydrous DMF to the flask and evaporated under reduced pressure toremove any trace of methanol present in the dendrimer which could impactthe efficiency of Steglich esterification. After the evaporation of DMF,the flask was brought under nitrogen environment. Added anhydrous DMF(10 mL/gram) to the flask and shifted the solution on sonication bathand sonicated the reaction mixture until clear solution was achieved.5-Hexynoic acid was dissolved in 2 ml of DMF and added to the stirringsolution. After 10 minutes, EDC.HCl and DMAP were added to the stirringsolution and the solution was left on stirring at room temperature for48 hours. On completion, DMF dialysis was started in 1 kDa molecularweight cut-off dialysis bag. DMF dialysis was performed for 8 h,replacing DMF once. After 8 h, 30 mL of D.I. water was added to thesolution in the bag and was dialysed against water for overnight. Thereaction mixture was diluted with HPLC water and the final volume wasmade 300-350 mL. TFF was performed in D.I water using 3 kDa TFFcartridge. The TFF cycle was performed 6-7 times and the final retentatevolume was around 100 mL which was lyophilized to obtained sticky solid.The product yield was around 5.5 g (74%). The ¹H NMR was recorded at 500MHz instrument in deuterated DMSO where 10 mg of compound was used forsample preparation. The loading of hexynoic acid was calculated byproton integration method. The internal amide peak of dendrimer between68.11-7.70 ppm was the reference peak. The peak at δ4.0 ppmcorresponding to ester linked protons and peak at 61.6 ppm is the CH₂from hexynoic acid. Proton integration method suggested the attachmentof 9-10 molecules of hexynoic acid per dendrimer. The HPLC purity was>99%.

TABLE 1 Reagents for Synthesis of dendrimer hexyne (Compound 2 of FIG.6A) 5-Hexynoic G4-OH acid EDC.HCl DMAP M.W 14279 112.13 191.7 122.17Amount   10.5 g  1.15 g  3.52 g  1.79 g Millimoles:   0.73  10.3  18.4 14.7 Equivalents   1  14  25  20

Synthesis of Dendrimer-Didesethyl Sunitinib Conjugate (Compound 3 inFIG. 6B)

Placed dendrimer hexyne (compound 2 of FIG. 6A) in the 250 mL roundbottom flask. Dissolved the compound in 40 mL anhydrous DMF bysonication. The sunitinib-azide solution was added in the reactionmixture by dissolving it in 20 mL DMF and solution was stirred. It wasfollowed by the addition of 10 mL of water to the reaction mixture tostop the precipitation of copper salt in reaction mixture. After 10minutes of stirring, copper sulfate pentahydrate (dissolved in 3 mL ofwater) was added dropwise to the reaction flask. The stirring solutionturned blue in color. After 5 minutes, sodium ascorbate (dissolved in 3mL of water) was added dropwise to the reaction mixture and the reactionvial was shifted over oil bath which was set at 40° C. The reactionmixture was stirred and heated for 24 h. On completion, the DMF wasevaporated and the reaction mixture was diluted with 300 mL of 10% DMAcin water. To this solution, EDTA (500 microliter, 0.5M) solution wasadded for copper salt removal by chelation. TFF was performed on thereaction mixture in water using 3 kDa TFF cartridge. 8-10 Diavolumeswere performed in 10% DMAc in water, followed by 5-6 cycles in water asbuffer to remove the solvent traces. The final retentate volume wasaround 150 mL which was lyophilized to obtain off yellow solid. Theproduct yield was 5.5 g. The ¹H NMR was recorded at 500 MHz instrumentin deuterated DMSO and deuterated water and around 10 mg of compound wasused for sample preparation. 100 scans were made for the ¹H NMR. The ¹HNMR indicated the formation of product and 6-7 arms of sunitinibmolecule were attached (FIG. 4). The drug loading is calculated byproton integration method where peaks corresponding to dendrimer anddrug are compared. The CH₂ peak at 1.8 ppm corresponding to hexynoicacid and ester linked CH₂ at 4.0 ppm is locked as the reference peakfrom dendrimer side. After the triazole formation, the new peak in ¹HNMR corresponding to CH₂ peak protons next to triazole ring at δ 4.4ppm, 2 aromatic protons from sunitinib in between 6.92-6.80 ppm and 2 NHprotons at 10.9 and 13.6 ppm were used to calculate the loading of drugmolecules. After the click reaction, there is formation of 1-4 triazoleand the signature proton peak corresponding to triazole appears inbetween δ 7.5-8.0 ppm which is suppressed by the presence of internalamide protons. For confirmation of sunitinib attachment, the ¹H NMR wasrecorded in D₂O where the disappearance of internal amide peaks andpresence of triazole peak at δ 7.7 ppm were observed. The HPLC puritywas >99%.

TABLE 2 Reagents for Synthesis of Dendrimer-didesethyl SunitinibConjugate (D-4517) Desethyl Compound 2 sunitinib-azide CuSO₄.5H₂O Naascorbate M.W ~15000 571.6 249.69 168 mg Amount     5.1 g  1.65 g 170 mg250 mg Millimoles:     0.340  2.89  0.68  0.85 Equivalents     1  8.5  2 2.5

Protocol for the In Vitro Kinase Binding Assay

Kinase-tagged T7 phage strains were prepared in an E. coli host derivedfrom the BL21 strain. E. coli were grown to log-phase and infected withT7 phage and incubated with shaking at 32° C. until lysis. The lysateswere centrifuged and filtered to remove cell debris. The remainingkinases were produced in HEK-293 cells and subsequently tagged with DNAfor qPCR detection. Streptavidin-coated magnetic beads were treated withbiotinylated small molecule ligands for 30 minutes at room temperatureto generate affinity resins for kinase assays. The liganded beads wereblocked with excess biotin and washed with blocking buffer (SEABLOCK®(Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand andto reduce non-specific binding. Binding reactions were assembled bycombining kinases, liganded affinity beads, and test compounds in 1×binding buffer (20% SEABLOCK®, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Testcompounds were prepared as 111× stocks in 100% DMSO. Kds were determinedusing an 11-point 3-fold compound dilution series with three DMSOcontrol points. All compounds for Kd measurements are distributed byacoustic transfer (non-contact dispensing) in 100% DMSO. The compoundswere then diluted directly into the assays such that the finalconcentration of DMSO was 0.9%. All reactions performed in polypropylene384-well plate. Each was a final volume of 0.02 ml. The assay plateswere incubated at room temperature with shaking for 1 hour and theaffinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). Thebeads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20,0.5 μM non-biotinylated affinity ligand) and incubated at roomtemperature with shaking for 30 minutes. The kinase concentration in theeluates was measured by qPCR.

Sample Preparation:

Sunitinib malate, sunitinib ester amide linker and D4-sunitinibconjugate were dissolved in aqueous DMSO to form solution at free drug(sunitinib) concentration of 10 mM. Each sample solution was furtherdiluted to 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.123 μM, 41.2 nM, 13.7 nM,4.57 nM, 1.52 nM, 0.508 nM and 0.169 nM in DMSO respectively.

Results

Synthesis and Characterization:

The synthesis of dendrimer-didesethyl sunitinib analogue is achieved in3 steps via copper(I) catalysed alkyne-azide click reaction (FIGS. 6Aand 6B). The first step involves the partial modification of thedendrimer surface hydroxyl groups by attaching a few hexynoic acidlinker arms by esterification to bring alkyne surface groups. Secondstep is the introduction of a linker arm on the didesethyl sunitinibwith azide terminal. Third step involves the click reaction of bothparts. For the synthesis of dendrimer hexynoic acid, the as-receivedethylenediamine core hydroxyl polyamidoamine dendrimer (G4-OH, Pharmagrade>95% HPLC purity) was used. Partial esterification of the OHterminated dendrimer (compound 1) was first performed (FIG. 6A) with5-hexynoic acid using the EDC.HCl and DMAP in anhydrous N,N-dimethylformamide to yield compound 2. ¹H NMR was used for theconfirmation of the compound. The loading of the hexyne linker wascalculated by proton integration method by comparing the linker protonsto the internal amide protons of the dendrimer between 68.11-7.70 ppm.The peak at 64.0 ppm corresponding to ester linked protons and peak at61.6 ppm refers the CH₂ from hexynoic acid. Proton integration methodsuggested the attachment of 8-10 molecules of alkyne linker perdendrimer. The purity of the construct was evaluated using HPLC and itwas found to be >99%. Linker 3-[2-(2-propoxyethoxy)ethoxy]propanoic acidwas attached to didesethyl sunitinib using EDC.HCl, DMAP, DIPEA, HOBt inanhydrous DMF to bring azide to participate in click reaction. The drugis connected through the linker with an amide linkage. The dendrimerhexyne (2) and didesethyl sunitinib PEG azide were used for performingclick reaction. Copper catalysed click reaction is one of the mostefficient chemical transformation which has brought revolution in thefield of drug discovery and is an excellent tool for conjugation ofsmall or big molecules to macromolecules, polymers and antibodies. It isknown for its easy execution, milder reaction conditions, compatibilitywith different functional groups, regio-selective, enhanced reactionrates, produce cleaner products with great yields. Copper (II) sulphatepentahydrate and sodium ascorbate were used for the click reaction inthe presence of DMF:H₂O (1:1). The reaction was carried out at roomtemperature for overnight followed by the purification by tangentialflow filtration. The formation of product (3) was confirmed by ¹H NMR.The ¹H NMR spectrum of the conjugate clearly shows the peakscorresponding to the dendrimer, drug and linkers attached to it, and thedrug loading was calculated by comparing these peaks with the help ofproton integration method. The internal amide protons from the dendrimerare present in between δ 8.5-7.5 ppm when spectrum is recorded indeuterated DMSO. These amide peaks are a reference standard for the restof the peaks. The —NH peaks from drug appear at δ 13.6 and 10.8 ppm.There are 4 protons from the drug and one triazole proton which isformed after the click reaction merged with internal amide peaks andcomes in between δ 8.5-7.5 ppm. Additionally, two aromatic protons fromsunitinib situated next to the fluorine group appear at δ 6.95-6.85 ppm.A sharp triazole peak at δ 7.7 ppm which is a signature peak for theclick transformation is observed when the NMR solvent is switched fromdeuterated DMSO to CD₃OD. After the click, the CH₂ present next to theazide downshielded and can be observed at δ 4.4 ppm. The comparison ofproton NMR spectra of drug linker, dendrimer intermediate and the finalconjugate was confirmed by ¹H NMR. The purity of the dendrimer drugconjugate, intermediate and drug linker was evaluated using HPLC. Thefinal conjugate is >99% pure by HPLC. The dendrimer G4-OH and dendrimerhexyne intermediate is visible at 210 nm channel and the didesethylsuntinib is visible at 430 nm in HPLC. The retention time of thecompound 2 is around 16.9 minutes but once the hydrophobic drugmolecules are attached to the dendrimer, the peak of the final conjugateshifts towards the right and comes around 27 minutes which confirms theattachment of hydrophobic drugs to the dendrimer construct. Once thedrug is attached to dendrimer the peak corresponding to it at both 210nm (dendrimer absorption wavelength) and 430 nm (drug absorptionwavelength) channels was observed, which further confirms the formationof product. The drug loading of the dendrimer conjugate is around 12.6%wt/wt which corresponds to 7 molecules of drug attached per dendrimermolecule.

Binding Affinity:

The kinase comparative binding affinity ofD-didesethyl-Sunitinib-conjugate (Compound D-4517), free sunitinibmalate and sunitinib linker (AVT-4517) was evaluated, and the resultsare presented in Table 3. The binding affinity for free sunitinib is0.13 nM. After the attachment of PEG linker, the binding affinitydecreased around 8 folds to 1.0 nM. The conjugate exhibited the bindingaffinity of 27 nM. The results demonstrate that the conjugation of drugon the dendrimer surface retains the binding affinity of the drug to RTKdomain in nanomolar range. This shows that the conjugate itself isactive and can bind to the receptor without the release of the drug. Ithas been shown for the first time that conjugation of a small moleculeinhibitor (300-400 Da) to a large dendrimer (14000 Da) can still retainnanomolar binding of the small molecule inhibitor.

TABLE 3 D-didesethyl-Sunitinib-conjugate Kinase (VGEFR2) binding assaystudy: Gene Kd Compounds Structure Symbol (nM) Sunitinib Malate

VEGFR2 0.13 Sunitinib ester amide linker (AVT-4517)

VEGFR2 1.0 D- didesethyl- Sunitinib- conjugate (D-4517)

VEGFR2 27

Stability Studies in Human and Rat Plasma:

In vitro stability of D-didesethyl sunitinib (D-4517) in human and ratplasma was further evaluated at physiological conditions. The resultspresented in Table 4 suggest that the conjugate D4517 is very stablewith 2% (weight percentage) release in human plasma, and 4% (weightpercentage) release in rat plasma) after 48 h.

TABLE 4 In vitro stability study of drug release percentage ofD-didesethyl sunitinib (D-4517) by weight in human and rat plasma at 37°C. Release percentage in Release percentage in Time point (hours) humanplasma (%) rat plasma (%) 0.5 0.13 0.15 1 0.12 0.17 2 0.16 0.31 4 0.20.59 6 0.28 0.81 8 0.54 1.42 24 0.76 1.97 48 2.01 3.98

In Vitro Drug Release Study:

In vitro drug release study was carried out at pH 7.4 and pH 5.5 withesterases at 37° C. mimicking plasma and intracellular conditionsrespectively. The release study was carried out in duplicates. Theresults are presented in FIG. 7. At intracellular conditions, less than2 wt % drug arm is being released in 15 days. At plasma conditions, ˜2%is being released in 24 h and around ˜4% in 15 days. The ester bondbetween the dendrimer and the linker resulted in a loss of the linkerwith AVT-4517 over time in D-4517. At both conditions, the conjugatedemonstrated good stability.

Example 6: In Vivo Pharmacokinetics of Dendrimer-Didesethyl SunitinibConjugate (D-4517)

D-4517 pharmacokinetics were evaluated in vivo in mice. Female C57/B16mice were injected I.P. with 5 or 50 mg/kg D-4517 and blood samples werecollected for determination of plasma D-4517 concentrations. Peak plasmaconcentrations were observed at the first time point sampled, 0.5 h.Exposure based on Cmax and AUC was dose related and approximately doseproportional. The terminal elimination, T_(1/2), was about 1 hourfollowing both dose levels. PK parameters estimated by noncompartmentalmethods are shown in Table 5 below and mean plasma concentrations versustime are shown in FIG. 8.

TABLE 5 Plasma D-4517 Concentrations Following IP Injection in Mice.Cmax AUC0-t AUCINF CL/F Dose (μg/ Tmax (h*μg/ (h*μg/ (mL/h/ Vz/F T1/2mg/kg mL) (h) mL) mL) kg) (mL/kg) (h) 5.0 6.86 0.50 7.70 11.7 426 6751.10 5.0 108 0.50 122 130 384 578 1.04

Toxicokinetic data was collected in rats. Sprague-Dawley rats receiveddaily I.P. injections of 12 mg/kg or a single dose of 168 mg/kg D-4517or a daily oral dose of 30 mg/kg sunitinib (40.21 mg/kg of sunitinibmalate). Blood samples were collected, and plasma drug concentrationswere determined. Noncompartmental toxicokinetic parameters wereestimated.

FIGS. 9A and 9B show plasma concentration versus time profiles on Day 1and Day 14 for the 12 mg/kg D-4517 and sunitinib groups. There did notappear to be an obvious gender difference in D-4517 plasmaconcentrations, but for sunitinib, males had higher concentrations thanfemales except at the 24 h time point on day 1. D-4517 plasmaconcentrations showed a faster decline than sunitinib. Terminalhalf-lives could not be reliably estimated because there were not enoughtime points in the terminal phase. Sunitinib was measurable 24 hoursafter dosing but D-4517 was not measurable beyond 8 hours post-dosing.Consequently, AUC estimates were higher for sunitinib compared toD-4517. Following a single dose of 168 mg/kg D4517, plasmaconcentrations were high at 1 h post dosing but at the next time pointsampled, 24 h, only one animals had a measurable concentration.

The pharmacokinetic results indicate that the dose of D-4517 resulted incomparable maximum concentrations with lower total exposure compared tosunitinib. Separate rats received sunitinib malate orally for 14 days ata dose of 40.21 mg/kg. D-4517 was associated with no mortality noreffects upon clinical observations, body weights, food consumption orclinical pathology parameters (hematology, clinical chemistry andurinalysis). D-4517-related gross necropsy findings were limited toyellow discoloration of adipose tissue and mesentery in males andfemales at 12 mg/kg and/or 168 mg/kg, which correlated withsubacute/chronic inflammation associated with intraperitonealadministration of test article. Organ weight changes includedstatistically significant decreases in spleen weights in males at 168mg/kg though this observation had no microscopic correlate. Non-adversemicroscopic findings included of minimal focal pigment in the choroid ofthe eyes and subacute/chronic inflammation in the abdominalfat/mesentery in males and females at 12 mg/kg and 168 mg/kg.Inflammation was likely secondary to intraperitoneal injection of testarticle and was observed secondarily along the serosal surfaces of thestomach, liver, and spleen.

Overall, D-4571 was well tolerated following single or repeated IPdosing. These observations were in contrast to sunitinib malate whichwas associated with various clinical and pathological changes inaddition to mortality in female rats.

Example 7: Single Subcutaneous Dosing Study of Dendrimer-DidesethylSunitinib Conjugate (D-4517)

To evaluate the preferred route of dosing in humans, a singlesubcutaneous dosing study of D-4517 was conducted in the laser-inducedCNV mouse model. Control mice (n=8/group) were injected intravitreallywith either vehicle or aflibercept (40 μg) one day after lasertreatment. Three dose levels of D-4517 (2, 10 and 50 mg/kg; n=8/group)was administered as a single subcutaneous dose one day after lasertreatment. After 14 days, mice were sacrificed and flat mounts of thesclera-choroid/RPE complexes were stained with DAPI and isolectin 1B4.CNV area was measured with fluorescent microscopy and imaging software.As shown in FIG. 10, all three doses of D-4517 given as a singlesubcutaneous dose reduced CNV lesion area significantly. The responsesin the D-4517 treated animals were more consistent than those observedin the aflibercept treated animals. This study demonstrates significantefficacy observed from subcutaneously administered D-4517 in CNV models.

Example 8: Conjugation of Didesethyl Sunitinib Via a Non-Cleavable EtherLinkage on Dendrimer

Methods

Synthesis of Dendrimer Conjugate Via a Non-Cleavable Ether Linkage onDendrimer

The synthesis began by the construction of a bifunctional dendrimer. Atdendrimer generation 3.5, 7 alkyne functional groups were introducedusing a polyethyl glycol (PEG) linker with an amine at one end and ahexyne at the other end to produce a generation 4 bifunctional dendrimer(compound 1 in FIG. 11) with 7 alkyne arms and 57 hydroxyl groups on thesurface. The structure of the dendrimer was confirmed by 1H NMRspectroscopy.

The clickable didesethyl sunitinib analog (compound 2 in FIG. 11, a.k.a.AVT-4517), including of didesethyl sunitinib, a three ethylene glycol(PEG3) spacer and a terminal azide, was synthesized to participate inthe click reaction with alkyne groups on the surface of the dendrimer.The active agent, compound 2, is manufactured using a 5-step synthesisshown in FIG. 5.

AVT-4517 (compound 2 in FIG. 11) is finally reacted with thebifunctional dendrimer (compound 1 in FIG. 11) with hexyne groups bycopper (I) catalyzed alkyne-azide click chemistry to yield D-4517.2(compound 3 in FIG. 11) with the full structure shown in FIG. 12. Afterconjugation of the analog to the dendrimer, the D-4517.2 is purified bytangential flow filtration (TFF) to remove any impurities and enablepurification into the final formulation.

¹H-NMR Analysis of D-4517.2 Conjugates

The formation of product D-4517.2 is confirmed by 1H NMR. The 1H NMRspectrum of the conjugate clearly shows the peaks corresponding to thedendrimer, drug and linkers attached to it, and the drug loading wascalculated by comparing these peaks with the help of proton integrationmethod. The internal amide protons from the dendrimer are present inbetween δ 8.5-7.5 ppm when spectrum is recorded in deuterated DMSO.These amide peaks are a reference standard for the rest of the peaks.The —NH peaks from drug appear at δ 13.6 and 10.8 ppm. There are 4protons from the drug and one triazole proton which is formed after theclick reaction merged with internal amide peaks and comes in between δ8.5-7.5 ppm. Additionally, 2 aromatic protons from sunitinib situatednext to the fluorine group appear at δ 6.95-6.85 ppm. A sharp triazolepeak at δ 7.7 ppm which is a signature peak for the click transformationis observed when the NMR solvent is switched from deuterated DMSO toCD₃OD. After the click, the CH₂ present next to the azide down shieldedand can be observed at δ 4.4 ppm. NMR is also used to quantitate thenumber of drug molecules conjugated to the hydroxyl dendrimer. The drugloading was calculated by proton integration method by comparing theprotons of dendrimer internal amide protons to drug protons.

HPLC Analysis for Assessment of Purity of D-4517.2

The purity of the dendrimer drug conjugate, intermediate and drug linkerwas evaluated using HPLC. The final conjugate is >99% pure by HPLC. Thedendrimer G4-OH and dendrimer hexyne intermediate is visible at 210 nmchannel and the didesethyl suntinib is visible at 430 nm in HPLC. Theretention time of the compound 2 is around 16.9 minutes but once thehydrophobic drug molecules are attached to the dendrimer, the peak ofthe final conjugate shifts towards the right and comes around 27minutes, which confirms the attachment of hydrophobic drugs to thedendrimer construct. Once the drug is attached to dendrimer the peakcorresponding to it can be observed at both 210 nm (dendrimer absorptionwavelength) and 430 nm (drug absorption wavelength) channels, whichfurther confirms the formation of product. The drug loading of thedendrimer conjugate is around 12.6% wt/wt which corresponds to 7molecules of drug attached per dendrimer molecule.

Size and Zeta Potential

The size and the zeta potential distribution of the D-4517.2 aredetermined using a Zetasizer Nano ZS instrument. For the sizemeasurement, the sample was prepared by dissolving the dendrimer indeionized water (18.2Ω) to make a solution with a final concentration of0.5 mg/mL. The solution was then filtered through 0.2 m syringe filters(Pall Corporation, 0.2 m HT Tuffryn membrane) directly into the cell (UVtransparent disposable cuvette, Dimensions: 12.5×12.5×45 mm). For zetapotential measurement, the sample was prepared at a concentration of 0.2mg/mL in 10 mM NaCl using above mentioned procedure. Malvern ZetasizerNanoseries disposable folded capillary cell was used for themeasurements. The size of D-4517 was 5.5±0.5 nm and zeta potential wasslightly positive (+5.4±0.4 mV).

Size Exclusion Chromatography Multiple-Angle Laser Scattering (SEC-MALS)

The molar mass of D-4517.2 will be determined by size exclusionchromatography multipleangle laser scattering (SEC-MALS).

Results

D-4517 has nanomolar affinity for VEGFR2 and does not require therelease of the active drug, AVT-4517. To further increase the stabilityof the conjugate under physiological conditions and further reduce therelease of the drug from the conjugate as observed in D-4517 buffer andplasma stability studies, the cleavable ester linkages on the dendrimersurface were replaced with non-cleavable linkages as demonstrated in thestructure of D-4517.2 (FIG. 12). There are no cleavable bonds in thestructure of D-4517.2.

D-4517.2 is a covalent conjugate of generation-4, hydroxyl-terminatedPAMAM dendrimers, containing an ethylene diamine (EDA) core, amidoaminerepeating units [CH₂CH₂CONHCH₂CH₂N]), and 64 hydroxyl end groups(chemical formula: C₆₂₂H₁₁₈₄N₁₈₆O₁₈₈) with didesethyl sunitinib analog(AVT-4517) conjugated to the dendrimer by a highly efficient clickchemistry approach. The hydroxyl, generation-4, PAMAM dendrimers aremono-disperse and produced with high compositional purity (>95%). Forthe preparation of D-4517.2, seven of the 64 hydroxyl groups on thedendrimer are modified to attach AVT-4517 (˜12.6% of total mass).

Stability Studies in Human, Mouse and Rat Plasma

In vitro stability of dendrimer didesethyl sunitinib conjugates, D-4517and D-4517.2, in human, mouse and rat plasma was evaluated atphysiological conditions. The results presented in FIG. 13. Comparedwith D4517 (2% (weight percentage) release in human plasma, and 4%(weight percentage) release in rat plasma), the plasma stability ofD4517.2 is improved significantly. At 48 hrs, in all three plasma, lessthan 0.5% drug (by weight) was released from dendrimer drug conjugates.

Binding Affinity

The kinase comparative binding affinity of D-4517 and D-4517.2 wasevaluated, and the results are presented in Table 6.

TABLE 6 Dendrimer didesethyl sunitinib conjugates, D-4517 and D-4517.2binding assay study Gene Compound Name Symbol Modifier Kd (nM) D4517VEGFR2 = 27 D4517 VEGFR1 = 1100 D4517 CSF1R = 82 D4517 KIT = 3.4 D4517PDGFRA = 16 D4517 PDGFRB = 11 D-4517.2 CSF1R = 41 D-4517.2 VEGFR1 = 890D-4517.2 KIT = 3 D-4517.2 PDGFRA = 11 D-4517.2 PDGFRB = 7.5 D-4517.2VEGFR2 = 14

The IC50 results of D-4517.2 is lower than D-4517 on all tested assay,which indicates the stronger binding between D4517.2 and tyrosine kinasereceptor.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

24. A dendrimer of formula (I):

wherein, D is a poly(amidoamine) (PAMAM) dendrimer selected fromgeneration 2, generation 3, generation 4, generation 5, generation 6,generation 7, generation 8, generation 9, and generation 10, L is one ormore linking moieties or spacers, X is an active agent or a derivative,analog or prodrug thereof, n is an integer from 1 to 100, m is aninteger from 16 to 4096, and Y is a linker selected from secondaryamides (—CONH—), tertiary amides (—CONR—), sulfonamide (—S(O)₂—NR—),secondary carbamates (—OCONH—; —NHCOO—), tertiary carbamates (—OCONR—;—NRCOO—), carbonate (—O—C(O)—O—), ureas (—NHCONH—; —NRCONH—; —NHCONR—,—NRCONR—), carbinols (—CHOH—, —CROH—), disulfide groups, hydrazones,hydrazides, and ethers (—O—), wherein R is an alkyl group, an arylgroup, or a heterocyclic group.
 25. The dendrimer of claim 24, wherein,D is a poly(amidoamine) (PAMAM) dendrimer selected from generation 4,generation 5, generation 6, generation 7, and generation 8, L is one ormore linking or spacer moieties, X is an inhibitor of vascularendothelial growth factor receptor (VEGFR), or an inhibitor of TIE2receptor tyrosine kinases, n is an integer from 1 to 100, m is aninteger from 16 to 4096; and Y is a bond or linkage that is minimallycleavable in vivo.
 26. The dendrimer of claim 24, wherein D is a G4PAMAM dendrimer, L is one or more linking or spacer moieties, X issunitinib, or a derivative, analog or prodrug thereof, Y is a secondaryamide (—CONH—), n is an integer from 5 to 15, m is an integer from 49 to59, and n+m=64.
 27. The dendrimer of claim 24, wherein D is a G4 PAMAMdendrimer, L is polyethylene glycol with a triazole linker, X is N,N-didesethyl sunitinib, or a derivative, analog or prodrug thereof, Y isa secondary amide (—CONH—), n is an integer from 5 to 15, m is aninteger from 49 to 59, and n+m=64.
 28. The dendrimer of claim 24,wherein formula I has the following structure:


29. The dendrimer of claim 28, wherein formula I has the followingstructure:


30. The dendrimer of claim 24, wherein the dendrimer has a diameter offrom about 1 nm to about 20 nm.
 31. The dendrimer of claim 30, whereinthe dendrimer has a diameter of from about 2 nm to about 10 nm.
 32. Thedendrimer of claim 24, wherein the dendrimer has a surface chargebetween −20 mV and 20 mV, between −10 mV and 10 mV, between −10 mV and 5mV, between −5 mV and 5 mV, or between −2 mV and 2 mV, inclusive. 33.The dendrimer of claim 24, wherein about 0.1% to about 40% of the totalsurface groups of the dendrimer are covalently linked to active agent oran analog thereof.